RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/669,1 18 filed May 9, 2018, the contents of which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure provides yeast that are capable of producing RNA bioactive molecules, including RNA interference molecules. Also provided are methods and uses of the yeast for delivering RNA bioactive molecules to a subject in need thereof.

BACKGROUND

[0003] The world population is expected to grow by 38% to 11 .2 billion by the end of the century and there is an urgent need to make sure that the number of people who are undernourished decreases from the 1.1 billion reported in 2009 (Butler, 2010). While agricultural output after the Green Revolution currently outpaces human consumption, this balance is likely unsustainable, especially as arable land is converted to residential, commercial and industrial properties and the shift in growing economically high-value but low efficiency crops and livestock, such as strawberries and beef.

[0004] To that end, the world applies roughly 6 million tons of pesticides (worth US$ 56 billion) and over 3,000 tons of antibiotics (worth US$

17.9 billion) annually (Atwood & Paisley, 2017; Pagel & Gautier, 2012; Van

Boeckel, Brower, & Gilbert, 2015). The major goal of these pesticides and antibiotics is to allow more intensive agricultural practices such as mono- culturing and combine harvesting for crops and feedlot farming for livestock.

However even with the integration of pesticides and antibiotics, roughly 40% of crop productivity and 18% of livestock productivity is lost worldwide due to agricultural pests and diseases (Drummond, Lambert, & Smalley, 1981 ; Oerke, 2006).

[0005] In terms of pesticides, part of this is due to intrinsically inaccurate administration; for example, the most common way to apply pesticides is through aerial spraying (crop dusting) and it is estimated that only 0.003 to 0.0000001 % of applied pesticides ever reaches its intended target pest (Pimentel & Burgess, 2012), leaving the bulk (99.99%) to impact the surrounding environment and food chain. Furthermore, for pesticides applied aerially, roughly 50-70% never even reaches the ground, instead becoming“spray drift” which then effects surrounding non-farm areas such as forests and rivers (Pimentel, 2005).

[0006] Antibiotics are also not without their downsides as well. Since the wide spread use of antibiotics in livestock started in the 1940s, more and more cases of reduced antibiotic efficacy have appeared, even at ever increasing dosages due to microorganisms adapting and gaining resistances. A prime example is Salmonella enterica Typhimurium DT104, originally an exotic bird disease that has now become an epidemic in cows, pigs, chickens and humans.

[0007] Wide spread use of pesticides and antibiotics has also had significant unintended consequences on the environment and species biodiversity. Indeed, pesticide run off has been found to decrease biodiversity in streams by 42% in Europe and 27% in Australia, with susceptible species including insects, fish, crustaceans and birds (Beketov & Kefford, 2013). Indirect routes of antibiotic transmission into the environment have also been discovered through antibiotic-laced livestock waste contaminating the water supply, including lakes and rivers used to water crops, thus effecting the environment and the human food supply chain at the same time.

RNA Interference [0008] Given the aforementioned negative environmental and health- related effects of pesticides and antibiotics, as well as the need for a continually expanding food supply to keep pace with the growing population, there is an unmet need for novel agricultural bio-control technologies. Indeed, innovations such as genetically modified crops, integrated crop management, biological pest control (i.e. insect predators), probiotics, plasmid vaccination, and RNA interference (RNAi) have been explored or implemented. Of these technologies, RNAi is an attractive technology as it is organism specific, non- toxic to the environment, and potentially immune to resistance.

[0009] The concept of using RNAi as a bio-control agent is not in itself new; in addition to winning the Nobel Prize in 2006, Andrew Fire and Craig Mello were also issued a patent (US 6,506,559 B1 ) for RNAi which included “a method to inhibit expression of a target gene in an invertebrate organism...” (Fire, Kostas, Montgomery, & Timmons, 2003). It is also well established that exogenous RNAi effector molecules can be administered by genetic engineering (direct or vector mediated) or through environmental applications, such as soaking, injection, and/or feeding, depending on the target organism (Joga, Zotti, & Smagghe, 2016). However, significant biological, commercial and technical limitations to RNAi and the US 6,506,559 B1 patent in particular have made it difficult to use in commercial agriculture applications; current commercial application of RNAi in agricultural bio-control is generally directed towards specific targets modulating existing bio-control methodologies (such as knocking out Bt resistance in pest organisms) rather than as a platform in itself.

SUMMARY

[0010] The present inventors have demonstrated that modification of one or more key gene regulators of RNA production, regulation and degradation have significantly improved the expression of heterologous RNA but not RNA generally. This disclosure has a wide range of applications including the fields of crop bio-pesticides, bio-control of invasive species, livestock and aquaculture disease prophylaxis and/or treatment and as a therapeutic for human diseases.

[0011] Accordingly, the present disclosure provides a yeast cell comprising an RNA instability gene(s) that is downregulated or inactivated and/or an RNA stability gene(s) that is upregulated or heterologously expressed; and at least one heterologous sequence that encodes an RNA bioactive molecule. In an embodiment, the heterologous sequence is integrated into the yeast genome. In another embodiment, the heterologous sequence is plasmid-based.

[0012] In an embodiment, the yeast is Saccharomyces, such as S. cerevisiae.

[0013] In an embodiment, the RNA bioactive molecule is an mRNA. In another embodiment, the RNA bioactive molecule is an RNAi effector molecule.

[0014] In one embodiment, the RNAi effector molecule is siRNA, miRNA, IhRNA, shRNA, dsRNA, or anti-sense RNA. In a particular embodiment, the RNAi effector molecule is dsRNA. In another embodiment, the RNAi effector molecule is long hairpin RNA (IhRNA).

[0015] The RNA stability gene may be any gene or combination of genes that increase production or stabilize RNA in the yeast. In an embodiment, two RNA stability genes are upregulated or heterologously expressed.

[0016] In an embodiment, the RNA stability gene is in an expression cassette that is integrated into the yeast genome. In another embodiment, the RNA stability gene is plasmid-based.

[0017] In an embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of CCR4 or THPI . In another embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of XRN1 or TAF1. [0018] The RNA instability gene may be any gene or combination of genes in the yeast that decrease production, destabilize or degrade RNA. In an embodiment, the RNA instability gene that is downregulated or inactivated comprises or consists of APN1 , DBR1 , DCS1 , EDC3, HBS1 , HTZ1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MKT 1 , MPP6, MRT4, NAM7, NMD2, PAP2, POP2, RNH1 , RNH203, RPS28A, RRP6, SIR3, SKI2, SKI3, SKI7, SKI8, SLH1 , TRF5, or UPF3. In one embodiment, the RNA instability gene comprises or consists of HBS1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MPP6, NAM7, NMD2, RRP6, SKI2, SKI3, or SKI7. In a particular embodiment, the RNA instability gene comprises or consists of LRP1. In another particular embodiment, the RNA instability gene comprises or consists of RRP6. In yet another particular embodiment, the RNA instability gene comprises or consists of SKI3. In a further particular embodiment, the RNA instability gene comprises or consists of MAK10. In yet a further particular embodiment, the RNA instability gene comprises or consists of MPP6.

[0019] In an embodiment, two RNA instability genes are downregulated or inactivated.

[0020] In one embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of RRP6 and SKI3. In another embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and RRP6. In yet another embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and MAK3. In a further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and SKI2. In yet a further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of SKI2 and SKI3. In an even further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of SKI3 and MAK3. [0021] In an embodiment, the RNA instability gene is downregulated or inactivated by deletion of the RNA instability gene in the yeast genome. In another embodiment, the RNA instability gene is downregulated or inactivated by any modification that reduces or abolishes its function, such as truncation, introduction of a stop codon or by point mutation. In yet another embodiment, the yeast may heterologously express factors that degrade or otherwise inactivate the protein product of the RNA instability gene (e.g. a dominant negative allele).

[0022] In an embodiment, the mRNA bioactive molecule encodes a protein that is useful for the treatment of a disease and/or infection, optionally immune factors that negatively regulate infection, such as stimulatory cytokines for macrophages; a protein that is related to a protein deficiency; or a protein that can elicit an immune or vaccine response for prevention or treatment of disease and/or infection.

[0023] In an embodiment, the RNAi effector molecule targets a gene involved in survival, maturation or reproduction of pests, or other infectious organisms, such as parasites, fungi, bacteria or viruses. In another embodiment, the RNAi effector molecule targets a gene involved in promoting a disease state, for example, in livestock, plants or humans.

[0024] In an embodiment, the gene involved in survival, maturation or reproduction comprises or consists of actin VATPase, cytochrome P450, hemolin, hunchback, bellwether, fez2, bicoid, modsp, boule, gas8, gnbpal , gnpba3, tubulin, Sad , Ire, otk or vitellogenin. In one embodiment, the gene involved in survival, maturation or reproduction comprises or consists of bellwether or fez2. In another embodiment, the gene involved in promoting a disease state comprises or consists of actin, VATPase, cytochrome p450, hemolin, hunchback, vitellogenin, VEGF, VEGFR1 , DDIT4, KRT6A, RRM2, p53, LMP2, LMP7, MECL1 , I L-1 b or TNF-a. In one embodiment, the disease is inflammatory bowel disease and the disease promoting gene is I L-1 b [0025] The present disclosure also provides a method of producing a yeast cell that produces an increased amount of RNA bioactive molecules, the method comprising downregulating or inactivating an RNA instability gene disclosed herein and/or upregulating or heterologously expressing an RNA stability gene disclosed herein; and expressing at least one heterologous sequence that encodes an RNA bioactive molecule disclosed herein. In an embodiment, the method comprises integrating the at least one heterologous sequence into the yeast genome. In another embodiment, the method comprises introducing at least one plasmid-based heterologous sequence into the yeast. In an embodiment, downregulating or inactivating the RNA instability gene comprises deleting the gene from the yeast genome. In another embodiment, downregulating or inactivating the RNA instability gene comprises modifying it to reduce or abolish its function, such as by truncation, introduction of a stop codon or by point mutation. In yet another embodiment, the yeast may heterologously express factors that degrade or otherwise inactivate the protein product of the RNA instability gene (e.g. a dominant negative allele).

[0026] Further provided herein is a method of biocontrol comprising exposing an unwanted organism to a yeast cell that produces increased amounts of an RNA bioactive molecule as disclosed herein, wherein the RNA bioactive molecule reduces the survival, maturation or reproduction of the unwanted organism.

[0027] In an embodiment, exposing the organism to the yeast cell comprises feeding the yeast cells to the unwanted organism, optionally fresh, semi-dry or dry yeast.

[0028] In one embodiment, the RNA bioactive molecule that reduces the survival, maturation or reproduction of the unwanted organism is an mRNA that encodes for a toxic factor or a negative regulatory factor in a host harboring the unwanted organism. [0029] In another embodiment, the RNA bioactive molecule is an RNAi effector molecule that targets a gene in the unwanted organism that is responsible for survival, maturation or reproduction. In an embodiment, the unwanted organism is a pest, a bacteria, a virus, a fungus or a parasite.

[0030] In one embodiment, the unwanted organism is an agricultural pest, such as an insect, and the RNAi effector molecule targets and silences the expression of at least one gene required by the pest for survival, maturation and/or reproduction. In an embodiment, the unwanted organism is a mosquito or a fly.

[0031] In an embodiment, the gene required by the pest is actin, VATPase, cytochrome p450, hemolin, hunchback, bellwether, fez2, bicoid, modsp, boule, gas8, gnbpal , gnpba3, tubulin, Sad , Ire, otk or vitellogenin. In one embodiment, the gene required by the pest is bellwether or fez2.

[0032] Even further provided herein is a method of treating a disease comprising exposing a subject having the disease to a yeast cell that produces increased amounts of an RNA bioactive molecule as disclosed herein, wherein the RNA bioactive molecule treats the disease.

[0033] In an embodiment, exposing the subject to the yeast comprises feeding the yeast cells to the subject, optionally as fresh, semi-dry or dry yeast. In another embodiment, exposing the subject to the yeast comprises intravenously, intradermally, intramuscularly, or subcutaneously injecting the yeast cell in the subject. In another embodiment, exposing the subject to the yeast comprises topical application or spraying of a solution of the yeast on the subject.

[0034] In one embodiment, the RNA bioactive molecule is an mRNA that encodes a protein that is useful for the treatment of the disease, optionally immune factors that negatively regulate infection, such as stimulatory cytokines for macrophages; an mRNA that encodes a protein that is related to a protein deficiency; or an mRNA that encodes a protein that can elicit an immune response for prevention or treatment of the disease.

[0035] In another embodiment, the RNA bioactive molecule is an RNAi effector molecule that targets a disease promoting gene in the subject.

[0036] In an embodiment, the subject is a plant or animal, such as livestock, a companion animal or a human.

[0037] In one embodiment, the disease promoting gene comprises or consists of VATPase, cytochrome p450, hemolin, hunchback, vitellogenin, VEGF, VEGFR1 , DDIT4, KRT6A, RRM2, p53, LMP2, LMP7, MECL1 , IL-1 b or TNF-a.

[0038] In one embodiment, the disease is inflammatory bowel disease and the disease promoting gene is I L-1 b.

[0039] Also provided is a method of treating an infection in a subject comprising exposing a subject having the infection to a yeast cell that produces increased amount of an RNA bioactive molecule as disclosed herein, wherein the RNA bioactive molecule is useful for treatment of the invention.

[0040] In one embodiment, the RNA bioactive molecule is an mRNA that encodes a protein that is useful for the treatment of the infection, optionally immune factors that negatively regulate infection, such as stimulatory cytokines for macrophages; or an mRNA that encodes a protein that can elicit an immune response for prevention or treatment of the infection.

[0041] In another embodiment, the RNA bioactive molecule is an RNAi effector molecule as disclosed herein, wherein the RNAi effector molecule targets an organism causing the infection in the subject or targets a host factor that promotes the infection in the subject. In an embodiment, the organism causing the infection is a virus, fungus, parasite or bacteria. [0042] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Embodiments are described below in relation to the drawings in which:

[0044] Figure 1A and Figure 1 B show a schematic representation of the RNAi effector reporter construct and expression vector. Figure 1A. Construct contains a RNAi effector stem loop sequence driven by the S. cerevisiae TEF1 promoter and CYC1 terminator signals. The construct also contains a nourseothricin resistance cassette (natMX6) and is flanked by trp1 homology arms for integration into the yeast genome. Figure 1 B. The reporter construct was then integrated into pRS423-KanMX expression vector using Gibson cloning.

[0045] Figure 2 shows RNAi expression profile screening of RNA processing gene knock out strains. Total RNA from various single gene knockout strains containing the RNAi reporter construct was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change relative to the wildtype yeast (white bar) and normalized to the reference gene ACT 1.

[0046] Figure 3 shows RNAi reporter expression profiles of select RNA processing single mutant strains. Total RNA from various single gene knockout strains containing the RNAi reporter construct was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change relative to the wildtype yeast (white bar) and normalized to the reference gene ALG1. Statistics were calculated using one-way ANOVA with WT sample as control; ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 , ^**** P < 0.0001 .

[0047] Figure 4 shows RNAi reporter expression profiles of select RNA processing double mutant strains. Total RNA from various double gene knockout strains, plus single gene knockout controls, containing the RNAi reporter construct was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change relative to the wildtype yeast (white bar) and normalized to the reference gene ALG1. Statistics were calculated using one-way ANOVA with WT sample as control; * P < 0.05, ** P < 0.01 , *** P < 0.001 , **** P < 0.0001 .

[0048] Figure 5 shows RNAi reporter expression profiles of candidate RNA stability gene single knockout mutants. Total RNA from various single gene knockout strains containing the RNAi reporter construct was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wildtype yeast (white bar) and normalized to the reference gene ALG9. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the wild type control: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001.

[0049] Figure 6 shows RNAi reporter expression profiles of XRN1 and TAF1 overexpressing strains. Total RNA from wild type BY4742 yeast cells bearing an RNAi effector expression construct integrated into the TRP1 locus and overexpressing XRN1 or TAFI was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wildtype yeast (white bar) and normalized to the reference gene 18S rRNA. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the wild type control: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 . [0050] Figure 7 shows a schematic representation of plasmid-based RNAi-effector expression construct.

[0051] Figure 8 shows RNAi reporter expression profiles of integrated vs. plasmid-based RNAi-effector expression constructs. Total RNA from integrated and plasmid-based RNAi-effector expression constructs in both BY4742 wild type and BY4742 Arrp6 ski3 cells was used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wild type, genome integrated reporter yeast (BY4742 TRP1 ::blw) and normalized to the reference gene ALG9. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the reference sample described above: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 .

[0052] Figure 9 shows a schematic representation of the RPR1 promoter-driven RNAi-effector expression construct. The RPR1 promoter- driven RNAi-effector expression construct was integrated into the yeast genome at the TRPI locus.

[0053] Figure 10 shows a schematic representation of the SNR33 promoter-driven RNAi-effector expression construct. The SNR33 promoter- driven RNAi-effector expression construct was maintained episomally on a 2- micron yeast plasmid, pRS343.

[0054] Figure 11 shows RNAi reporter expression profiles of gene knockout strains bearing a genome-integrated RPR1 promoter-driven RNAi- effector expression construct. Total RNA from BY4742 wild type and BY4742 gene-knockout cells, all containing an RPR1 promoter-driven RNAi-effector expression construct integrated into the TRP1 locus, were used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wild type yeast (white bar) and normalized to the reference gene ALG9. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the reference sample described above: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 .

[0055] Figure 12 shows RNAi reporter expression profiles of low and high copy plasmids bearing an SNR33 promoter-driven RNAi-effector expression construct. Total RNA from BY4742 wild type cells transformed with either low or high copy plasmids containing either a TEF1 promoter- driven or SNR33 promoter-driven RNAi-effector expression construct were used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wild type yeast and normalized to the reference gene ALG9. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the reference sample described above: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001.

[0056] Figure 13 shows RNAi reporter expression profiles of different RNAi effector constructs. Total RNA from BY4742 wild type cells and BY4742 Arrp6/Aski3 cells transformed with either genome-integrated or plasmid-based TEF1 promoter-driven RNAi-effector expression constructs were used as input for quantitative reverse transcriptase PCR. Reporter gene expression is given as fold change (mean) relative to the wild type yeast transformed with a single copy TRP1 locus integrated RNAi effector expression construct and normalized to the reference gene ALG9. Error bars represent standard error of the sample mean between triplicate samples. Statistics were calculated using two-tailed T-test between each individual sample and the reference sample described above: ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001.

[0057] Figure 14 shows survival of D. melanogaster adults during feeding trials with S. cerevisiae expressing hairpin RNA against bellwether (blw) (SEQ ID NO: 33). D. melanogaster adults were fed ad libitum with yeast expressing blw-dsRNA. The number of live adults in each vial was determined at each timepoint, and percentage survival values were calculated relative to the day 2 live flies. Values represent the means and standard deviations of 3 replicates vials, each containing 20 adult flies at time zero. Error bars represent 1 standard error of the mean.

[0058] Figure 15 shows survival of Ae. aegypti larvae during feeding trials with S. cerevisiae hairpin RNA against fez2 (SEQ ID NO: 34). Survival of Aedes aegypti larvae fed on agar pellets containing yeast expressing fez2- dsRNA. The number of larvae after 24 h was determined, to correct for deaths due to handling injuries, and percentage survival values were calculated relative to the day 1 survivors. Values represent the means and standard errors of 4-6 replicates, starting with 40 larvae at time zero. Error bars represent 1 standard error of the mean.

[0059] Figure 16A and Figure 16B show LhRNA targeting I L-1 b (SEQ ID NO: 41 ) reduced histological evidence of disease in SHIP deficient mice. 6- week-old SHIP deficient mice were treated with either yeast containing IhRNA or control yeast. Ileal cross-sections were fixed and stained with H&E and histological damage was scored in SHIP deficient mice after 10 days (Figure 16A) or 14 days (Figure 16B). N=4 mice per group in total.

[0060] Figures 17A, 17B and 17C show LhRNA targeting I L-1 b (SEQ ID NO: 41 ) reduced disease activity and histological damage in DSS-treated Malt1- ^A mice. Maitl^ mice were subjected to 2% DSS for 6 days and were treated with either yeast containing IhRNA targeting I L-1 b or control yeast. (Figure 17A) Disease activity index was measured daily in mice during DSS treatment. (Figure 17B) Colon cross-sections were fixed and stained with H&E and histological damage was scored. (Figure 17C) Survival rate (>15% weight loss = humane end point) was calculated for Maltl^ mice. N = 4 mice / group for one experiment.

DETAILED DESCRIPTION

[0061] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0062] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term“consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

[0063] As used herein, the singular forms“a”, “an” and“the” include plural references unless the content clearly dictates otherwise. The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes the degree of error associated with measurement of the particular quantity). When referring to a period such as a year or annually, it includes a range from 9 months to 15 months. All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.

[0064] The term“heterologous” as used herein refers to a sequence that is foreign to the host yeast cell.

[0065] The term “integrated” sequence as used herein refers to the foreign sequence being inserted into the host yeast genome. Yeast

[0066] The present disclosure provides a yeast cell comprising an RNA instability gene that is downregulated or inactivated and/or an RNA stability gene that is upregulated or heterologously expressed; and at least one heterologous sequence that encodes an RNA bioactive molecule. In an embodiment, the yeast cell comprises an RNA instability gene that is downregulated or inactivated or an RNA stability gene that is upregulated or heterologously expressed. In another embodiment, the yeast cell comprises an RNA instability gene that is downregulated or inactivated and an RNA stability gene that is upregulated or heterologously expressed.

[0067] In one embodiment, the at least one heterologous sequence is integrated into the yeast genome. In a particular embodiment, the at least one heterologous sequence is integrated at the trp locus. In another embodiment, the at least one heterologous sequence is present in the yeast in a plasmid.

[0068] In an embodiment, the at least one heterologous sequence comprises a constitutively active promoter for expressing the RNA bioactive molecule. In another embodiment, the at least one heterologous sequence comprises an inducible promoter for expressing the RNA bioactive molecule. In an embodiment, the at least one heterologous sequence comprises an RNA pol II promoter, such as an RNA pol II constitutively active promoter, for example TEF1 . In another embodiment, the at least one heterologous sequence comprises an RNA pol III promoter, such as an RNA pol III constitutively active promoter, for example RPR1 or SNR33.

[0069] In an embodiment, the yeast is a food-grade yeast. In one embodiment, the yeast is from Saccharomyces. In a particular embodiment, the yeast is Saccharomyces cerevisiae.

[0070] In an embodiment, the yeast comprises at least one RNA stability gene that is upregulated or heterologously expressed and at least one RNA instability gene that is downregulated or inactivated. [0071] The RNA stability gene may be any gene from any source that is involved in the production or stabilization of RNA in the yeast, such that upregulation or heterologous expression of said gene results in increased production or stabilization of RNA in the yeast, compared to a yeast where the RNA stability gene is not upregulated or heterologously expressed.

[0072] In an embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of CCR4 or THPI . In another embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of XRN1 or TAF1.

[0073] The term “CCR4” as used herein refers to CCR4 or Carbon Catabolite Repression 4 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof. S. cerevisiae CCR4 has the nucleic acid sequence as shown in Genbank Gene ID: 851212 or

Saccharomyces Genome Database (SGD) No: S000000019 or NCBI

Reference Sequence: NM_001 178166.1 . The term “THP1” as used herein refers to THP1 or Tho2/Hpr1 Phenotype that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof. S. cerevisiae THP1 has the nucleic acid sequence as shown in Genbank Gene ID: 854082 or Saccharomyces Genome Database (SGD) No: S000005433 or NCBI

Reference Sequence: NM_001 183327.1 .

[0074] The term “XRN1” as used herein refers to XRN1 or eXoRiboNuclease 1 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof. S. cerevisiae XRN1 has the nucleic acid sequence as shown in Genbank Gene ID: 852702 or

Saccharomyces Genome Database (SGD) No: S000003141 or NCBI

Reference Sequence: NM_001 181038.1 . The term “TAF1” as used herein refers to TAF1 or TATA binding protein-Associated Factor 1 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof. S. cerevisiae TAF1 has the nucleic acid sequence as shown in Genbank Gene ID: 853191 or Saccharomyces Genome Database (SGD) No: S000003506 or NCBI Reference Sequence: NM_001 181403.2.

[0075] In an embodiment, the yeast comprises two RNA stability genes that are upregulated or heterologously expressed. In another embodiment, the yeast comprises three RNA stability genes that are upregulated or heterologously expressed. In a further embodiment, the yeast comprises four RNA stability genes that are upregulated or heterologously expressed. In yet a further embodiment, the yeast comprises 5, 6, 7, 8 or more RNA stability genes that are upregulated or heterologously expressed.

[0076] In an embodiment, the yeast comprises an expression cassette that is optionally integrated in its genome that codes for the RNA stability gene. In another embodiment, the yeast comprises a plasmid that codes for the RNA stability gene.

[0077] The yeast may be used to produce increased quantities of the RNA bioactive molecule or RNA bioactive molecules compared to a yeast where the RNA stability gene (or genes) has not been upregulated or heterologously expressed. In an embodiment, the production is increased by at least 1.25-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 2000-fold or more.

[0078] The RNA instability gene may be any gene in the yeast that is involved in degradation or destabilization of RNA, such that downregulation or inactivation of said gene results in increased production or stabilization of RNA or decreased degradation of RNA, compared to a yeast where the RNA instability gene is not downregulated or inactivated.

[0079] In an embodiment, the RNA instability gene that is downregulated or inactivated comprises or consists of APN1 , DBR1 , DCS1 , EDC3, HBS1 , HTZ1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MKT 1 , MPP6, MRT4, NAM7, NMD2, PAP2, POP2, RNH1 , RNH203, RPS28A, RRP6, SIR3, SKI2, SKI3, SKI7, SKI8, SLH1 , TRF5, or UPF3. In one embodiment, the RNA instability gene comprises or consists of HBS1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MPP6, NAM7, NMD2, RRP6, SKI2, SKI3, or SKI7. In a particular embodiment, the RNA instability gene comprises or consists of LRP1. In another particular embodiment, the RNA instability gene comprises or consists of RRP6. In yet another particular embodiment, the RNA instability gene comprises or consists of SKI3. In a further particular embodiment, the RNA instability gene comprises or consists of MAK10. In yet a further particular embodiment, the RNA instability gene comprises or consists of MPP6.

[0080] The term “APN1” as used herein refers to APN1 or DNA- (apurinic or apyrimidinic site) lyase APN1 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof. S. cerevisiae APN1 has the nucleic acid sequence as shown in Genbank Gene ID: 853746 or Saccharomyces Genome Database (SGD) No: S000001597 or NCBI Reference Sequence: NM_001 179680.1 . The term “DBR1” as used herein refers to DBR1 or RNA lariat debranching enzyme that may be from any yeast source, for example, S. cerevisiae or homologs thereof. S. cerevisiae DBR1 has the nucleic acid sequence as shown in Genbank Gene ID: 853708 or SGD No. S000001632 or NCBI Reference Sequence: NM_001 179715.1 . The term “DCS1” as used herein refers to DCS1 or 5'-(N(7)-methyl 5'- triphosphoguanosine)-(mRNA) diphosphatase that may be from any yeast source, for example, S. cerevisiae or homologs thereof. S. cerevisiae DCS1 has the nucleic acid sequence as shown in Genbank Gene ID: 850974 or SGD No. S000004260 or NCBI Reference Sequence: NM_001 182157.1 . The term “EDC3” as used herein refers to EDC3 or Enhancer Of mRNA DeCapping that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae EDC3 has the nucleic acid sequence as shown in Genbank Gene ID: 856700 or SGD No. S000000741 or NCBI Reference Sequence: NM_001 178830.1 . The term “HBS1” as used herein refers to HBS1 or ribosome dissociation factor GTPase HBS1 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae HBS1 has the nucleic acid sequence as shown in Genbank Gene ID: 853959 or SGD No. S000001792 or NCBI Reference Sequence: NM_001 179874.3. The term ΉTZ1” as used herein refers to HTZ1 or histone H2AZ that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae HTZ1 has the nucleic acid sequence as shown in Genbank Gene ID: 854150 or SGD No. S000005372 or NCBI Reference Sequence: NM_001 183266.1 . The term ΊRK1” as used herein refers to IPK1 or inositol pentakisphosphate 2-kinase that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae IPK1 has the nucleic acid sequence as shown in Genbank Gene ID: 851910 or SGD No. S000002723 or NCBI Reference Sequence: NM_001 180623.3. The term“LRP1” as used herein refers to LRP1 or Like RrP6 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae LRP1 has the nucleic acid sequence as shown in Genbank Gene ID: 856481 or SGD No. S000001 123 or NCBI Reference Sequence: NM_001 17921 1 .1 . The term “MAK3” as used herein refers to MAK3 or peptide alpha-N-acetyltransferase MAK3 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MAK3 has the nucleic acid sequence as shown in Genbank Gene ID: 856163 or SGD No. S000006255 or NCBI Reference Sequence: NM_001 184148.1 . The term “MAK10” as used herein refers to MAK10 or Maintenance of Killer 10 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MAK10 has the nucleic acid sequence as shown in Genbank Gene ID: 856657 or SGD No. S000000779 or NCBI Reference Sequence: NM_001 178868.3. The term “MAK31” as used herein refers to MAK31 or Maintenance of Killer 31 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MAK31 has the nucleic acid sequence as shown in Genbank Gene ID: 850383 or SGD No. S000000614 or NCBI Reference Sequence: NM_001 178734.1 . The term “MKT1” as used herein refers to MKT1 or Maintenance of K2 Killer Toxin that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MKT1 has the nucleic acid sequence as shown in Genbank Gene ID: 855639 or SGD No. S000005029 or NCBI Reference Sequence: NM_001 182923.3. The term “MPP6” as used herein refers to MPP6 or M-Phase Phosphoprotein 6 homolog that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MPP6 has the nucleic acid sequence as shown in Genbank Gene ID: 855758 or SGD No. S000005307 or NCBI Reference Sequence: NM_001 183201.3. The term“MRT4” as used herein refers to MRT4 or mRNA Turnover 4 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae MRT4 has the nucleic acid sequence as shown in Genbank Gene ID: 853860 or SGD No. S000001492 or NCBI Reference Sequence: NM_001 179575.1 . The term“NAM7” as used herein refers to NAM7 or ATP- dependent RNA helicase NAM7 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae NAM7 has the nucleic acid sequence as shown in Genbank Gene ID: 855104 or SGD No. S000004685 or NCBI Reference Sequence: NM_001 182579.1 . The term “NMD2” as used herein refers to NMD2 or Nonsense-mediated MRNA Decay may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae NMD2 has the nucleic acid sequence as shown in Genbank Gene ID: 856476 or SGD No. S000001 1 19 or NCBI Reference Sequence: NM_001 179207.1. The term “PAP2” as used herein refers to PAP2 or non-canonical poly(A) polymerase PAP2 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae PAP2 has the nucleic acid sequence as shown in Genbank Gene ID: 854034 or SGD No. S000005475 or NCBI Reference Sequence: NM_001 183369.1 . The term“POP2” as used herein refers to POP2 or CCR4-NOT core DEDD family RNase subunit POP2 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae POP2 has the nucleic acid sequence as shown in Genbank Gene ID: 855788 or SGD No. S000005335 or NCBI Reference Sequence: NM_001 183229.3. The term“RNH1” as used herein refers to RNH1 or RNA-DNA hybrid ribonuclease that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae RNH1 has the nucleic acid sequence as shown in Genbank Gene ID: 855274 or SGD No. S000004847 or NCBI Reference Sequence: NM_001 182741.1 . The term“RNH203” as used herein refers to RNH203 or Rnh203p that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae RNH203 has the nucleic acid sequence as shown in Genbank Gene ID: 850847 or SGD No. S000004144 or NCBI Reference Sequence: NM_001 182041 .1 . The term“RPS28A” as used herein refers to RPS28A or ribosomal 40S subunit protein S28A that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae RPS28A has the nucleic acid sequence as shown in Genbank Gene ID: 854338 or SGD No. S000005693 or NCBI Reference Sequence: NM_001 183586.1 . The term “RRP6” as used herein refers to RRP6 or exosome nuclease subunit RRP6 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae RRP6 has the nucleic acid sequence as shown in Genbank Gene ID: 854162 or SGD No. S000005527 or NCBI Reference Sequence: NM_001 183420.1 . The term“SIR3” as used herein refers to SIR3 or chromatin-silencing protein SIR3 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SIR3 has the nucleic acid sequence as shown in Genbank Gene ID: 851 163 or SGD No. S000004434 or NCBI Reference Sequence: NM_001 182330.3. The term “SKI2” as used herein refers to SKI2 or SKI complex RNA helicase subunit SKI2 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SKI2 has the nucleic acid sequence as shown in Genbank Gene ID: 851 1 14 or SGD No. S000004390 or NCBI Reference Sequence: NM_001 182286.3. The term “SKI3” as used herein refers to SKI3 or SKI complex subunit tetratrico peptide repeat protein SKI3 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SKI3 has the nucleic acid sequence as shown in Genbank Gene ID: 856319 or SGD No. S000006393 or NCBI Reference Sequence: NM_001 184286.1 . The term“SKI7” as used herein refers to SKI7 or Superkiller 7 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SKI7 has the nucleic acid sequence as shown in Genbank Gene ID: 854243 or SGD No. S000005602 or NCBI Reference Sequence: NM_001 183495.1 . The term “SKI8” as used herein refers to SKI8 or SKI complex subunit WD repeat protein SKI8 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SKI8 has the nucleic acid sequence as shown in Genbank Gene ID: 852659 or SGD No. S000003181 or NCBI Reference Sequence: NM_001 181078.1. The term“SLH1” as used herein refers to SLH1 or putative RNA helicase that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae SLH1 has the nucleic acid sequence as shown in Genbank Gene ID: 853187 or SGD No. S000003503 or NCBI Reference Sequence: NM_001181400.4. The term “TRF5” as used herein refers to TRF5 or non-canonical poly(A) polymerase TRF5 that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae TRF5 has the nucleic acid sequence as shown in Genbank Gene ID: 855417 or SGD No. S000005243 or NCBI Reference Sequence: NM_001 183137.1 . The term“UPF3” as used herein refers to UPF3 or UP Frameshift that may be from any yeast source, for example S. cerevisiae or homologs thereof. S. cerevisiae UPF3 has the nucleic acid sequence as shown in Genbank Gene ID: 852963 or SGD No. S000003304 or NCBI Reference Sequence: NM_001 181201.1 .

[0081] The term“homolog” as used herein refers to the same gene in a related species such as the same gene in a different yeast strain. Typically homologs share a high degree of sequence identity, such as at least 50%, 60%, 70% or more. The homology between two genes that are derived from species which are more closely related is typically higher than from more distantly related species. [0082] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity=number of identical overlapping positions/total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. An optional, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402. Alternatively, PSI-BLAST can be used to perform an iterated search, which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another optional, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1 1 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0083] In an embodiment, the yeast comprises two RNA instability genes that are downregulated or inactivated.

[0084] In one embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of RRP6 and SKI3. In another embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and RRP6. In yet another embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and MAK3. In a further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of LRP1 and SKI2. In yet a further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of SKI2 and SKI3. In an even further embodiment, the RNA instability genes that are downregulated or inactivated in the yeast comprise or consist of SKI3 and MAK3.

[0085] In yet another embodiment, the yeast comprises three RNA instability genes that are downregulated or inactivated. In a further embodiment, the yeast comprises four RNA instability genes that are downregulated or inactivated. In yet a further embodiment, the yeast comprises 5, 6, 7, 8 or more RNA instability genes that are downregulated or inactivated.

[0086] In an embodiment, the yeast that comprises the RNA instability gene that is downregulated or inactivated comprises a genome where the RNA instability gene has been deleted. In another embodiment, the yeast that comprises the RNA instability gene that is downregulated or inactivated comprises a genome where the RNA instability gene is downregulated or inactivated by any modification that reduces or abolishes its function, such as by truncation, introduction of a stop codon or by point mutation. In yet another embodiment, the yeast may heterologously express factors that degrade or otherwise inactivate the protein product of the RNA instability gene (e.g. a dominant negative allele).

[0087] The yeast may be used to produce increased quantities of the RNA bioactive molecule or RNA bioactive molecules compared to a yeast where the RNA instability gene (or genes) has not been downregulated or inactivated. In an embodiment, the production is increased by at least 1.25- fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 2000-fold or more

[0088] The RNA bioactive molecule refers to any biologically active RNA molecule, from any source or organism. In one embodiment, the RNA bioactive molecule is an mRNA molecule for producing a protein. In another embodiment, the RNA bioactive molecule is an RNAi effector molecule for inducing an RNA interference response.

[0089] In an embodiment, the mRNA bioactive molecule encodes a protein that is useful for the treatment of a disease and/or infection, optionally immune factors that negatively regulate infection, such as stimulatory cytokines for macrophages; a protein that is related to a protein deficiency; or a protein that can elicit an immune response for prevention or treatment of disease and/or infection. Examples of mRNA that would be useful for therapy are known in the art, including without limitation, vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) (Trepotec et al. 2018), ornithine transcarbamylase (Prieve et al. 2018), glucose-6-phosphate (Roseman et al. 2018), and Influenza hemagluttinins, Ebola virus glycoprotein, RSV-F, Rabies virus glycoprotein, HIV-1 gag, HSV1 -tk, hMUT, hEPO, Bcl-2 and ACE-2 (Xiong et al. 2018), and SERPINA1 (Connolly et al. 2018).

[0090] In an embodiment, the RNAi effector molecule is siRNA, miRNA, IhRNA, shRNA, dsRNA, or anti-sense RNA. In one embodiment, the RNAi effector molecule is dsRNA. In another embodiment, the RNAi effector molecule is long hairpin RNA (IhRNA).

[0091] The terms“RNA interference,”“interfering RNA” or“RNAi” refer to single-stranded RNA or double-stranded RNA (dsRNA) that is capable of reducing or inhibiting expression of a target nucleic acid by mediating the degradation of mRNAs which are complementary to the sequence of the interfering RNA when the interfering RNA is in the same cell as the target gene. Interfering RNA may have substantial or complete identity to the target nucleic acid or may comprise a region of mismatch.

[0092] The term“antisense RNA” refers to a single stranded RNA that is complementary to messenger RNA and that hybridizes with the messenger RNA blocking translation into protein.

[0093] The term“long hairpin RNA” or“IhRNA” as used herein refers to a long inhibitor RNA that can be used to reduce or inhibit expression of a target nucleic acid by RNA interference. LhRNA are typically single stranded with secondary structure (hairpin) and longer than 60 nucleotides. Total length may be 1000 base pairs or more. [0094] The term "siRNA" or“siRNA oligonucleotide” refers to a short inhibitory RNA that can be used to reduce or inhibit nucleic acid expression of a specific nucleic acid by RNA interference.

[0095] The siRNA can be a duplex, a short RNA hairpin (shRNA) or a microRNA (miRNA).

[0096] Methods of designing specific nucleic acid molecules that silence gene expression and administering them are known to a person skilled in the art. For example, it is known in the art that efficient silencing is obtained with siRNA duplex complexes paired to have a two nucleotide 3' overhang. The siRNA can also be chemically modified to increase stability. For example adding two thymidine nucleotides and/or 2Ό methylation is thought to add nuclease resistance. Other modifications include the addition of a 2’-0-methyoxyethyl, 2’-0-benzyl, 2’-0-methyl-4-pyridine, C-allyl, O-allyl, O-alkyl, O-alkylthioalkyl, O-alkoxylalkyl, alkyl, alkylhalo, O-alkylhalo, F, NH2, ONH2, O-silylalkyl, or N-phthaloyl group (see U.S. Pat. No. 7,205,399; Kenski et al. Mol. Ther. Nucl. Acids 1 :1 -8 (2012); Behlke, Oligonucleotides 18:305- 320 (2008)). Other modifications include direct modification of the internucleotide phosphate linkage, for example replacement of a non-bridging oxygen with sulfur, boron (boranophosphate), nitrogen (phosphoramidate) or methyl (methylphosphonate). A person skilled in the art will recognize that other nucleotides can also be added and other modifications can be made. As another example deoxynucleotide residues (e.g. dT) can be employed at the 3’ overhang position to increase stability.

[0097] The RNAi effector molecule may be any RNAi effector molecule that targets a gene of interest. In an embodiment, the gene of interest is involved in survival, maturation or reproduction of an unwanted organism, such as a pest, parasite, bacterium, fungus or virus. In another embodiment, the gene of interest is involved in promoting a disease state in an organism.

[0098] In an embodiment, the yeast comprises at least two heterologous sequences that encode an RNA bioactive molecule, such that two different RNA bioactive molecules are produced. In another embodiment, the yeast comprises at least three, at least four, at least five or more heterologous sequences that encode an RNA bioactive molecule, such that different RNA bioactive molecules are produced in the yeast.

Methods

[0099] The present disclosure also provides a method of making a yeast cell that produces an increased amount of RNA bioactive molecules, the method comprising downregulating or inactivating an RNA instability gene(s) as disclosed herein or upregulating and/or heterologously expressing an RNA stability gene(s) as disclosed herein in the yeast; and expressing at least one heterologous sequence that encodes the RNA bioactive molecule. In an embodiment, the method comprises downregulating or inactivating an RNA instability gene as disclosed herein or upregulating or heterologously expressing an RNA stability gene as disclosed herein in the yeast. In another embodiment, the method comprises downregulating or inactivating an RNA instability gene as disclosed herein and upregulating or heterologously expressing an RNA stability gene as disclosed herein in the yeast. In an embodiment, at least one RNA stability gene is upregulated or heterologously expressed and at least one RNA instability gene is downregulated or inactivated.

[00100] In an embodiment, the method comprises integrating the heterologous sequence into the yeast genome, for example, at the trp locus. In another embodiment, the method comprises inserting a plasmid into the yeast that codes for the heterologous sequence.

[00101] In an embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of CCR4 or THPI . In another embodiment, the RNA stability gene that is upregulated or heterologously expressed comprises or consists of XRN1 or TAF1. [00102] In an embodiment, the RNA instability gene that is downregulated or inactivated comprises or consists of APN1 , DBR1 , DCS1 , EDC3, HBS1 , HTZ1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MKT 1 , MPP6, MRT4, NAM7, NMD2, PAP2, POP2, RNH1 , RNH203, RPS28A, RRP6, SIR3, SKI2, SKI3, SKI7, SKI8, SLH1 , TRF5, or UPF3. In one embodiment, the RNA instability gene comprises or consists of HBS1 , IPK1 , LRP1 , MAK10, MAK3, MAK31 , MPP6, NAM7, NMD2, RRP6, SKI2, SKI3, or SKI7. In a particular embodiment, the RNA instability gene comprises or consists of LRP1. In another particular embodiment, the RNA instability gene comprises or consists of RRP6. In yet another particular embodiment, the RNA instability gene comprises or consists of SKI3. In a further particular embodiment, the RNA instability gene comprises or consists of MAK10. In yet a further particular embodiment, the RNA instability gene comprises or consists of MPP6.

[00103] In an embodiment, the method comprises downregulating or inactivating two RNA instability genes and/or upregulating or heterologously expressing two RNA stability genes.

[00104] In one embodiment, the two RNA instability genes comprise or consist of RRP6 and SKI3. In another embodiment, the two RNA instability genes comprise or consist of LRP1 and RRP6. In yet another embodiment, the two RNA instability genes comprise or consist of LRP1 and MAK3. In a further embodiment, the two RNA instability genes comprise or consist of LRP1 and SKI2. In yet a further embodiment, the two RNA instability genes comprise or consist of SKI2 and SKI3. In an even further embodiment, the two RNA instability genes comprise or consist of SKI3 and MAK3.

[00105] In yet another embodiment, the method comprises downregulating or inactivating three RNA instability genes and/or upregulating or heterologously expressing three RNA stability genes. In a further embodiment, the method comprises downregulating or inactivating four RNA instability genes and/or upregulating or heterologously expressing four RNA stability genes. In yet a further embodiment, the method comprises downregulating or inactivating 5, 6, 7, 8 or more RNA instability genes and/or upregulating or heterologously expressing 5, 6, 7, 8 or more RNA stability genes.

[00106] In an embodiment, downregulating or inactivating the RNA instability gene comprises deleting the RNA instability gene or otherwise modifying the yeast to reduce or abolish its function, for example, by truncation, introduction of a stop codon or by point mutation. A person skilled in the art would readily understand how to make a deletion of a yeast gene. Briefly, gene deletions can be obtained by any mutation, or combination thereof, that result in the partial or complete loss of protein function. For example, suitable mutations may include, but are not limited to, loss of promoter activity, loss of RNA translation, protein truncation, amino acid substitution, loss of coding sequence, etc. Such mutations can be achieved through multiple means of genome modification including, but not limited to, replacing all or a portion of a gene with target DNA encoding the desired mutation via homologous recombination, CRISPR/Cas9 genome editing, or other forms of gene editing.

[00107] In an embodiment, heterologously expressing the RNA stability gene or genes comprises integrating an expression cassette comprising the heterologous RNA stability gene or genes into the yeast genome. Briefly, such a cassette would comprise the RNA stability gene operationally linked to promoter and terminator sequences suitable for driving expression of the RNA stability gene. Such promoter and terminator sequences are generally known to those skilled in the art and include, but are not limited to, TEF1 , PGK1 , TDH3, REV1 , RNR2, GAL1 , ADH1 , etc. The cassette may be integrated into the yeast genome through multiple means of genome modification including, but not limited to, homologous recombination, CRISPR/Cas9 genome editing, or other forms of gene editing. In another embodiment, heterologously expressing the RNA stability gene or genes comprises the use of a plasmid to drive expression of the gene expression cassette(s).

[00108] Accordingly, in an embodiment, the at least one heterologous sequence comprises a constitutively active promoter for expressing the RNA bioactive molecule. In another embodiment, the at least one heterologous sequence comprises an inducible promoter for expressing the RNA bioactive molecule. In an embodiment, the at least one heterologous sequence comprises an RNA pol II promoter such as an RNA pol II constitutively active promoter, for example TEF1. In another embodiment, the at least one heterologous sequence comprises an RNA pol III promoter, such as an RNA pol III constitutively active promoter, for example RPR1 or SNR33.

[00109] In yet another embodiment, the yeast may heterologously express factors that degrade or otherwise inactivate the protein product of the RNA instability gene (e.g. a dominant negative allele).

[00110] The yeast cells disclosed herein are useful as a continuous source or delivery system of RNA bioactive molecules for a variety of applications.

[00111] For example, RNA interference molecules have been shown to be an effective biocontrol agent. For example, bacteria have been used to deliver dsRNA to control insects (Zhu et al. , 2010; Whitten et al., 2016) and insect vectors of disease (Taracena et al., 2015). The potential of yeast to be used as a biocontrol agent for insects has also been shown in a number of recent publications. For example, common S. cerevisiae expressing shRNA targeting Drosophila suzukii— a major cause of crop loss in soft summer fruit including cherries, blueberries, grapes and apricots— was shown to reduce activity and reproductive fitness (Murphy et al. 2016, W02017106171A1 ).

More recently, common S. cerevisiae was also engineered as hosts for shRNA expression targeting various genes required for viability of mosquito larvae (Hapairai, Mysore, Chen, & Harper, 2017; Mysore, Hapairai, & Sun,

2017). In all cases common, unoptimized yeast were used, thus the systems were not optimized for dsRNA production and/or delivery. It follows that having a biological delivery system that produces increased amount of RNA may be useful for biocontrol.

[00112] Accordingly, herein provided is a method of biocontrol comprising exposing an unwanted organism to a yeast cell that produces increased amounts of a RNA bioactive molecule, such as mRNA encoding a toxic factor or a negative regulatory factor, or an RNAi effector molecule(s) as disclosed herein, wherein the bioactive molecule reduces the survival, maturation or reproduction of the unwanted organism, for example, an RNAi effector molecule(s) targets a gene in the unwanted organism that is responsible for survival, maturation or reproduction. In an embodiment, the unwanted organism is a pest, a bacterium, a virus, a fungus or a parasite.

[00113] In an embodiment, exposing the unwanted organism to the yeast cell comprises feeding the yeast cell to the unwanted organism , or feeding the yeast cell to a host organism harboring the unwanted organism (e.g. host organism infected with a bacterium, virus, fungus or parasite).

[00114] In one embodiment, the unwanted organism is an agricultural pest, such as an insect, and the RNAi effector molecule(s) targets and silences the expression of at least one gene required by the pest for survival, maturation and/or reproduction. For example, RNAi effector molecules have been known to target survival genes such as actin, VATPase and cytochrome P450 (Anderson, Sheehan, Eckholm, & Mott, 201 1 ; Chang, Wang, Regev- Yochay, Lipsitch, & Hanage, 2014; Jin, Singh, Li, & Zhang, 2015; X. Li, Zhang, & Zhang, 201 1 ; Lin, Huang, Liu, & Belles, 2017; Murphy, Tabuloc, Cervantes, & Chiu, 2016), maturation genes such as hemolin and hunchback (Yu, Liu, Huang, & Chen, 2016) and reproduction genes such as vitellogenin (Vg) (Ghosh, Hunter, & Park, 2017; Lu, Vinson, & Pietrantonio, 2009; Whitten, Facey, & Del Sol, 2016).

[00115] Accordingly, in one embodiment, the pest is a fly and the gene required by the pest for survival is bellwether (blw). Bellwether encodes a subunit of the mitochondrial ATP synthase complex involved in the final enzymatic step of the oxidative phosphorylation pathway (Jacobs et al. 1998). Moreover, bellwether expression is known to regulate Drosophila lifespan in male flies (Garcia et al. 2017).

[00116] In another embodiment, the pest is a mosquito and the gene required by the pest for survival is fez2. Fez2 encodes fasciculation and elongation protein zeta 2 ( fez2 ), which is an essential neuronal factor necessary for normal axonal bundling and elongation within axon bundles (Fujita et al. 2004). Moreover, fez2 knockdown has been shown to significantly decrease viability of mosquito larvae (Hapairai et al. 2017).

[00117] mRNA molecules and RNA interference molecules also have applications in the treatment of disease. Accordingly, also provided herein is a method of treating a disease comprising exposing a subject having the disease to a yeast cell that produces increased amounts of an RNA bioactive molecule(s) as disclosed herein, wherein the RNA bioactive molecule(s) is useful for treating the disease. Also provided herein is use of a yeast cell that produces increased amounts of an RNA bioactive molecule(s) as disclosed herein for treating a disease in a subject, wherein the RNA bioactive molecule(s) is useful for treating the disease. Further provided herein is use of a yeast cell that produces increased amounts of an RNA bioactive molecule(s) as disclosed herein in the preparation of a medicament for treating a disease in a subject, wherein the RNA bioactive molecule(s) is useful for treating the disease. Even further provided is a yeast cell that produces increased amounts of an RNA bioactive molecule(s) as disclosed herein for use in treating a disease in a subject, wherein the RNA bioactive molecule(s) is useful for treating the disease.

[00118] In an embodiment, the organism is an aquaculture species, livestock, a companion animal, a plant or a human or any other animal. In the case of livestock/aquaculture species and humans or any other animal, the yeast cell containing RNA bioactive molecules can be fed to the organism in either a live or inactivated form. Other routes of administration or use include intravenous, intradermal, intramuscular and subcutaneous injections as well as topical use or spraying of a solution containing the yeast.

[00119] In one embodiment, the RNA bioactive molecule is an mRNA that encodes a protein that is useful for the treatment of the disease, an mRNA that encodes a protein that is related to a protein deficiency or an mRNA that encodes a protein that can elicit an immune response for prevention or treatment of the disease.

[00120] In an embodiment, the mRNA bioactive molecule encodes a protein that is useful for the treatment of a disease and/or infection, a protein that is related to a protein deficiency or a protein that can elicit an immune response for prevention or treatment of disease and/or infection. Examples of mRNA that would be useful for therapy are known in the art, including without limitation, vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) (Trepotec et al. 2018), ornithine transcarbamylase (Prieve et al. 2018), glucose-6-phosphate (Roseman et al. 2018), and Influenza hemagluttinins, Ebola virus glycoprotein, RSV-F, Rabies virus glycoprotein, HIV-1 gag, HSV1-tk, hMUT, hEPO, Bcl-2 and ACE-2 (Xiong et al. 2018), and SERPINA1 (Connolly et al. 2018).

[00121] In another embodiment, the RNA bioactive molecule is an RNAi effector molecule that targets a disease promoting gene in the subject. In an embodiment, the disease is a disease affecting the gut of the subject. In an embodiment, exposing the subject to the yeast comprises feeding the yeast cells to the subject.

[00122] Once delivered to an organism, RNAi typically enters the organism’s cells via endosomes, RNAi effectors are released into the cell, and then proceed to downregulate target disease genes through commonly accepted functional RISC RNA-protein complexes readily known to those skilled in the art, thereby eliminating or protecting the organism from diseases or pests (Bradford et al. , 2017).

[00123] In an embodiment, the disease (e.g. bacterial, viral, fungal or other parasite) promoting gene that is targeted is selected from one of the following classes including, but not limited to, native disease genes required for replication and/or survival, native disease genes required for virulence, host genes required for disease state (e.g. host factors responsible for infection), or host genes preventing immune system clearance of the disease (e.g. host factors attenuating immune response to the disease), and host genes that promote disease state.

[00124] Actin, VATPase and cytochrome P450 have been shown to be genes involved in survival (Anderson et al., 201 1 ; Chang et al., 2014; Jin et al., 2015; Li et al. 201 1 ; Lin et al., 2017; Murphy et al., 2016). Accordingly, in one embodiment, the disease promoting gene is actin, VATPase or cytochrome p450.

[00125] Hemolin and hunchback have been shown to be genes involved in maturation (Yu, Liu, Huang, & Chen, 2016). Accordingly, in another embodiment, the disease promoting gene is hemolin or hunchback.

[00126] Vitellogenin has been shown to be a gene involved in reproduction (Ghosh et al., 2017; Lu et al., 2009; Whitten et al., 2016). Accordingly, in another embodiment, the disease promoting gene is vitellogenin.

[00127] VEGF, VEGFR1 , and DDIT4 have been shown to play a role in age-related macular degeneration (Tiemann & Rossi, 2009). Accordingly, in an embodiment, the disease promoting gene is VEGF, VEGFR1 , or DDIT4.

[00128] KRT6A has been shown to play a role in pachyonychia congenita (Tiemann & Rossi, 2009). Accordingly, in another embodiment, the disease promoting gene is KRT6A. [00129] RRM2 has been shown to play a role in solid tumour formation (Tiemann & Rossi, 2009). Accordingly, in another embodiment, the disease promoting gene is RRM2.

[00130] p53 has been shown to play a role in acute renal failure

(Tiemann & Rossi, 2009). Accordingly, in another embodiment, the disease promoting gene is p53.

[00131] LMP2, LMP7, and MECL1 have been shown to play a role in metastatic melanoma (Tiemann & Rossi, 2009). Accordingly, in another embodiment, the disease promoting gene is LMP2, LMP7, or MECL1.

[00132] TNF-a has been shown to play a role in colon inflammation (Laroui et al., 201 1 ). Accordingly, in another embodiment, the disease promoting gene is TNF-a.

[00133] I L-1 b is a pro-inflammatory cytokine, which is known to be a primary regulator of inflammation (Coccia et al. 2012). I L-1 b plays a key role in the development of IBD by activating multiple types of immune cells. Progression of intestinal inflammation in patients with IBD is associated with increased levels of I L-1 b production (Coccia et al. 2012). Accordingly, in another embodiment, the disease promoting gene is I L-1 b, for example, for treating IBD.

[00134] RNA bioactive molecules also have applications in fighting infection. Interfering RNA can target genes within an infectious organism in order to decrease the infectivity or survival of the infectious organism and mRNA molecules can encode proteins that are useful in treating the infection or proteins that elicit an immune response against the infection.

[00135] Accordingly, also provided herein is a method of treating or preventing an infection in a subject comprising exposing a subject having the infection, or susceptible to the infection, to a yeast cell that produces increased amount of an RNA bioactive molecule(s) as disclosed herein, wherein the RNA bioactive molecule(s) is useful for treating or preventing the infection. Further provided is use of a yeast cell that produces increased amount of an RNA bioactive molecule(s) as disclosed herein for treating or preventing an infection in a subject, wherein the RNA bioactive molecule(s) is useful for treating or preventing the infection. Even further provided is use of a yeast cell that produces increased amount of an RNAi effector molecule(s) as disclosed herein in the preparation of a medicament for treating or preventing an infection in a subject, wherein the RNA bioactive molecule(s) is useful for treating or preventing the infection. Also provided is use of a yeast cell that produces increased amount of an RNA bioactive molecule(s) as disclosed herein for use in treating or preventing an infection in a subject, wherein the RNA bioactive molecule(s) is useful for treating or preventing the infection.

[00136] In an embodiment, the yeast cell is exposed to the subject or used orally.

[00137] In an embodiment, the organism causing the infection is a virus, fungus, parasite or bacterium.

[00138] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00139] The following non-limiting examples are illustrative of the present disclosure:

EXAMPLES

Example 1 :

Reporter strain development [00140] To be able to assess the impact of yeast modifications on RNAi effector expression, a reporter RNAi reporter gene system was developed containing the constitutive strong promoter ( TEF1 ), short hairpin DNA sequence targeting D. suzukii tubulin (-200 bp stem sequences and 74 bp loop sequence), CYC1 terminator, NatMX resistance marker cassette and TRP1 flanking regions. This cassette was assembled in expression vector pRS423-KanMX using Gibson cloning (SEQ ID NO:1 ). All the fragments required for the Gibson reaction were PCR amplified (SEQ ID 3, 4, 5, 6, 7, 8) and purified except the backbone vector (digested by EcoRV) and reporter gene cassette (digested by Kpnl and Sail). The assembled 2.6 kb reporter system (SEQ ID NO:2) was harvested by restriction enzyme digestion (, Bst1107Z) followed by DNA gel purification. A schematic diagram of this construct is shown in Figure 1. The purified DNA fragment was used as donor DNA for integrating the reporter system into the TRP1 locus of the haploid laboratory S. cerevisiae strain Y7092 ( MAT alpha , can1 delta: :STE2pr- Sp_his5 lypldelta his3delta1 Ieu2delta0 ura3delta0 met15delta0 ) using CRISPR/Cas9 technology (DiCarlo, Norville, Mali, & Rios, 2013) or homologous recombination (SEQ ID NO: 9 and 10).

[00141] The RNAi reporter construct was integrated into the genome of a haploid S. cerevisiae laboratory strain (Y7092), after which the reporter- containing query strain was crossed to the Stanford Yeast Deletion Genome Project collection (Winzeler, Shoemaker, & Astromoff, 1999). Following re- isolation of stable haploid strains, a set of 5000+ yeast deletion mutants were obtained, each of which contained a singular copy of the RNAi reporter constructs.

[00142] Using the research literature on yeast RNA processing as a guideline, a set of 350+ gene knockouts were shortlisted to test for their ability to increase steady state levels of the RNAi reporter. From this list, the RNAi reporter expression, via RT-qPCR, was assayed. In brief, total RNA was isolated using the hot acidic phenol-chloroform extraction protocol (Kohrer & Domdey, 1991 ), purified, and reverse transcribed into cDNA. Quantitative PCR was performed using either ACT1 or ALG1 as housekeeping genes (SEQ ID NO: 28, 29, 30, 31 , 71 , 72). The 46 most interesting knockouts derived from the short list of gene ontology and genetic interaction information were screened for reporter expression (Figure 2).

[00143] The most promising candidates— those with fold change in reporter expression less than 0.5 or greater than 1.5— were then analyzed in technical triplicate and normalized to the wild type Y7092 with genome integrated reporter gene construct (Figure 3). A number of genes were identified that, when knocked out, resulted in statistically higher expression of the RNAi reporter construct. More specifically, disruption of RRP6, LRP1 , and/or MPP6 (all essential components of the nuclear ribonucleic exosome complex), as well as members of the SKI‘Super Killer’ (SKI2, SKI3, and SKI7) and MAK ‘Maintenance of Killer’ (MAK3, MAK10, MAK31 ) gene families resulted in > 1 .5-fold increases in reporter gene expression.

[00144] For select genes from this group (LRP1 , RRP6, SKI2, SKI3, MAK3), double mutant haploid strains were constructed in which each strain contained one copy of the RNAi reporter construct, as well as two gene knockouts (Figure 4). To generate the double mutants, hygromycin B resistance cassettes were amplified using primers (SEQ ID NO: 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25) flanked by 50bp target gene homologous sequence. The purified DNA cassettes were transformed into selected single mutants using PEG3350/LiAc (Gietz & Schiestl, 2007). The transformants were selected on YEG plates containing both G418 and hygromycin and then confirmed genetically using primers upstream of target gene and inside antibiotic resistance gene (SEQ ID NO: 26 and 27). As seen in Figure 3, one particular gene knockout combination (RRP6/SKI3) was observed that showed a > 4-fold increase in reporter gene expression compared to wildtype and was substantially higher than either of the single knockout constituents. [00145] In all experiments the effect of the gene knockouts on the expression of reporter construct appears to be specific, at least in part, to the reporter construct rather than affecting global transcription/RNA levels. This unexpected result, as evidenced by fold change in reporter gene expression > 1 (relative to the transcription of the reference gene ACT1/ALG1 ), suggests, without wishing to be bound by theory, that specific combinations of gene knockouts are useful for facilitating the high-level expression of heterologous RNA constructs, such as RNAi effectors.

Discussion

[00146] Taken together, the data shown in Figures 2, 3 and 4 indicate that the exploitation of gene knockouts can be used for the optimization of RNAi effector expression in yeast. In this way, it would be expected that such modifications could be purposed for the optimized expression of any RNAi effector sequence. Thus, such strain(s) would be platform strains applicable to any end use target including insects (e.g. agricultural pests, disease vectors), animals (e.g. livestock, aquaculture, and humans).

Example 2: RNA stability genes

[00147] To identify RNA stability genes, a candidate set of seven deletion mutants involved in RNA stability were screened, each containing a genome-integrated RNAi effector reporter construct. Strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol- chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR measuring RNAi effector reporter expression was performed using ALG9 as a housekeeping gene for normalization of data (SEQ ID NO: 30, 31 , 71 , 72). Results of the RT-qPCR screening are shown in Figure 5.

[00148] Briefly, all of the deletion strains exhibited reporter expression at levels < 50% (0.5x fold change), relative to a wild type strain containing the RNAi reporter construct. Of note, XRN1 showed the lowest levels of reporter gene expression at 26% of wild type (Figure 5). [00149] Based on this, XRN1 (which encodes a dual function mRNA stability factor) was chosen as an RNA stability gene for overexpression. In addition, TAF1 , which could not be screened as a deletion due to its essential nature, was chosen to be overexpressed. TAF1 encodes a subunit of core general transcription factors and promotes RNA polymerase II transcription initiation.

[00150] To test the effect of RNA stability gene overexpression on RNAi effector expression, each of XRN1 and TAF1 was expressed from a strong constitutive promoter in wild type cells bearing a genome-integrated RNAi effector reporter construct. Strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol-chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR was performed using 18S rRNA as a housekeeping gene (SEQ ID NO: 71 , 72, 73 74). Results of the RT-qPCR screening are shown in Figure 6.

[00151] Relative to wild type cells, cells overexpressing XRN1 and TAF1 expressed significantly more RNAi effector, with relative levels of the reporter increased by 2.27- and 2.1 1 -fold, respectively. These data confirm the role of XRN1 and TAF1 as RNA stability genes suitable for overexpression to increase levels of RNAi effector molecule expression in yeast.

Example 3: Plasmid based RNAi effector expression

[00152] RNAi effectors may be expressed from genome integrated constructs or episomal (non-chromosomal) constructs, e.g. plasmids. To demonstrate the utility of a plasmid-based RNAi effector expression construct, a plasmid containing the same RNAi-effector construct as in the genome integrated version (Figure 1A) was created, but carried on a high-copy (2 micron) plasmid instead (Figure 7) (SEQ ID NO: 44).

[00153] To test the plasmid-based RNAi-effector expression construct, as well as its interaction with previously identified RNA instability genes (RRP6 / SKI3) that, when knocked out, were able to significantly upregulate genome-integrated RNAi effector reporter gene expression, the plasmid- based RNAi-effector expression construct was transformed into both wild type and Arrp6/Aski3 cells. Resultant strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol-chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR was performed using ALG9 as a housekeeping gene (SEQ ID NO: 30, 31 , 59, 60). Results of the RT- qPCR screening are shown in Figure 8.

[00154] As shown in Figure 8, the Arrp6/Aski3 mutation significantly increased integrated reporter gene expression by 3.4-fold, relative to wild type cells. On the other hand, the plasmid-based system expressed 28.8-fold more RNAi-effector reporter, relative to wild type cells. Lastly, the Arrp6/Aski3 mutation synergistically increased expression of the plasmid-based reporter, by 420-fold and 14.6-fold compared to wild type cells with the integrated- construct and plasmid-construct, respectively (Figure 8).

Example 4: RNA Polymerase Ill-based expression

[00155] RNAi effectors may be expressed as different forms (e.g. siRNA, miRNA, dsRNA, shRNA, IhRNA, or anti-sense RNA). As such, RNAi effectors may be expressed from multiple different classes of cellular promoters, including RNA polymerase II promoters and RNA polymerase III promoters. As shown above, RNAi effectors may be expressed from both genome-integrated and plasmid-based RNA polymerase II promoter constructs (Figures 1-8) and RNA instability and stability gene modifications increase levels of RNAi effector expression (Figures 1 -8).

[00156] To demonstrate that RNAi effectors can also be expressed from

RNA polymerase III promoters, expression constructs with either the yeast

RPR1 (Figure 9) and SNR33 (Figure 10) promoters, both RNA polymerase

III promoters, were constructed driving expression of an RNAi effector sequence (SEQ ID NO: 45 and 46). Of note, the yeast RPR1 gene encodes the RNA component of the nuclear RNase P Ribonucleoprotein, while the yeast SNR33 promoter encodes a small nucleolar protein involved in rRNA processing. RPR1 refers to RNase P Ribonucleoprotein 1 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof, and has the nucleic acid sequence as shown in Genbank Gene ID: 9164884 or SGD No. S000006490 or NCBI Reference Sequence: NR_132166.1 . SNR33 refers to Small Nucleolar RNA 33 that may be from any yeast species or source, for example, S. cerevisiae or homologs thereof, and has the nucleic acid sequence as shown in Genbank Gene ID: 9164874 or SGD No. S000007298 or NCBI Reference Sequence: NR_132156.1 .

[00157] To test the RPR1 promoter-driven RNAi-effector expression construct, the construct was integrated into the yeast genome at the TRP1 locus. Using this reporter strain, a panel of 12 RNA instability and stability gene knockouts were screened in parallel with the wild type reporter strain to determine the effects of RNA stability/instability gene modifications on RPR1 promoter-driven RNAi-effector gene expression. Strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol-chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR was performed using ALG9 as a housekeeping gene (SEQ ID NO: 30, 31 , 71 , 72). As shown in Figure 11 , the RPR1 RNA polymerase III promoter was suitable for expression of the RNAi effector, as reflected by detectable gene expression in wild type cells bearing the reporter. Furthermore, previously identified RNA instability gene modifications (i.e. LRP1 and RRP6) significantly upregulated reporter gene expression, 1.81 - to 2.25-fold in this system, respectively (Figure 11 ).

[00158] To test the SNR33 promoter-driven RNAi-effector expression construct, both low and high copy plasmids bearing the SNR33 promoter- driven RNAi-effector expression construct were created. These plasmids were transformed into wild type yeast strains alongside low and high copy plasmids with the previously characterized TEF1 promoter-driven RNAi-effector expression construct. Strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol-chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR was performed using ALG9 as a housekeeping gene (SEQ ID NO: 30, 31 , 71 , 72). As shown in Figure 12, in both the low and high copy plasmid constructs, the SNR33 promoter-driven RNAi-effector was able to be expressed to levels approximately 50% of that of the TEF1 promoter-driven RNAi-effector. This indicates that, like the RPR1 promoter, the SNR33 promoter is capable of expressing RNAi-effectors in yeast.

Example 5: Applicability to multiple, distinct RNAi effectors

[00159] Having demonstrated that RNAi effector genes can be expressed in yeast from either wild type or RNA instability/stability mutant cells (Figures 1-7), and from either a genome integrated or plasmid-based expression construct (Figure 8), next the capacity of these systems to support high-level expression of a variety of different, biologically-relevant RNAi effectors was tested.

[00160] To do so, TEF1 promoter-driven RNAi-effector expression constructs were constructed for the following genes: bicoid (SEQ ID NO: 32) and bellwether (SEQ ID NO: 33) from Drosophila melanogaster, fez2 (SEQ ID NO: 34), gas8 (SEQ ID NO: 35), gnbpal (SEQ ID NO: 36), gnbpa3 (SEQ ID NO: 37), boule (SEQ ID NO: 38), and modsp (SEQ ID NO: 39) from Aedes aegypti, and IL1B-1 (SEQ ID NO: 40), IL1B-2 (SEQ ID NO: 41 ), and IL1B-3 (SEQ ID NO: 42) from Mus musculus, as well as EGFP (SEQ ID NO: 43). Using these constructs, both genome-integrated and plasmid-based versions were created that were then transformed into either wild type yeast cells or Arrp6/Aski3 mutant yeast cells. All strains were screened by RT-qPCR, in which total RNA was extracted by hot acidic phenol-chloroform, purified, and reverse transcribed into cDNA. Quantitative PCR for each RNAi effector gene was performed using ALG9 as a housekeeping gene (SEQ ID 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 30, 31 ). [00161] As shown in Figure 13, for all RNAi effector genes, the

Arrp6/Aski3 mutant cells resulted in significantly higher levels of RNAi effector gene expression, relative to wild type cells. This was true for both genome- integrated and plasmid-based RNAi effector expression constructs. Across all RNAi effector genes, the average increase in expression level was 4.3- and 9.74-fold for the genome-integrated and plasmid-based expression constructs, respectively.

Example 6: Feeding based, biological activity in insects: Drosophila melanogaster

[00162] To test the insecticidal activity of the yeast-RNAi effector production and delivery system in an insect model system, D. melanogaster was used in a feeding and survival assay. Importantly D. melanogaster is a model organism for Dipteran insects and is closely related to a number of pest species, including Drosophila suzukii (Spotted Wing Drosophila) which is a fruit crop pest and is a serious economic threat to soft summer fruit (e.g. cherries, blueberries, raspberries, blackberries, peaches, nectarines, apricots, grapes, and others). D. suzukii has also been studied in the past with regards to RNAi based biocontrol (Murphy et al. 2016).

[00163] To induce an insecticidal effect in D. melanogaster , yeast expressing RNAi effectors targeting the D. melanogaster gene bellwether (blw) were developed (SEQ ID NO: 33). Bellwether encodes a subunit of the mitochondrial ATP synthase complex involved in the final enzymatic step of the oxidative phosphorylation pathway. It is an important protein involved in general energy metabolism and its knockdown was expected to have a detrimental effect in flies.

Materials and methods

Fly maintenance [00164] The wildtype D. melanogaster strain used in this study was Canton-S. The flies were reared in standard cornmeal media at 25 °C under non-crowded conditions.

Fly feeding experiments

[00165] Starvation media (SM - 5% sucrose, 2% agarose) laced with yeast paste expressing RNAi targeting bellwether was used for the survival assay. Each vial had 5 ml_ of food and approximately 200 uL of yeast paste. Three different yeast strains were used in the experiment: BY4742 Arrp6/Aski3, BY4742 plasmid-blw, and BY4742 Arrp6/Aski3 plasmid-blw. For each treatment, 3 replicate vials of flies were used. For all the treatments, 20 black pupae were placed in each experimental vial containing starvation media (SM) and yeast paste (pupae were picked from food vials raised in standard cornmeal media). The flies eclosed (emerging as adults) within 24 hours from being placed in vials were flipped into fresh vials with SM and yeast. They were flipped every two days and the number of flies dying in each treatment was noted until their numbers sufficiently dwindled. The flies were always flipped into food vials with starvation media laced with the appropriate yeast paste. All the experiments were conducted in a humidity-controlled incubator at 25 °C in 12-hour lightdark cycle environment. Of note, fresh yeast paste was used at each stage to maintain consistency in yeast RNAi effector levels. Starvation media was also made fresh every time the flies were flipped.

Results

[00166] Adult D. melanogaster were fed ad libitum for 18 days with the following yeast strains, BY4742 Arrp6/Aski3, BY4742 plasmid-blw, and

BY4742 Arrp6/Aski3 plasmid-blw. The number of live adults was determined at each timepoint (every 2 days), and percentage survival values were calculated relative to the starting point. As shown in Figure 14, compared to the negative control not expressing blw RNAi (BY4742 Arrp6/Aski3), both the yeast expressing blw RNAi (BY4742 plasmid-blw and BY4742 Arrp6/Aski3 plasmid-blw) induced an insecticidal effect in the flies. Comparing the wildtype yeast expressing blw RNAi and the Arrp6/Aski3 modified yeast expressing blw RNAi, we observed a substantial increase in insecticidal activity with < 10 % survival in BY4742 Arrp6/Aski3 plasmid-blw treated flies after day 14 (as compared to nearly 60% survival in BY4742 plasmid-blw treated flies at this timepoint). At the end of the study (day 18), we observed < 3% survival in BY4742 Arrp6/Aski3 plasmid-blw treated flies, compared to 32% survival in BY4742 plasmid-blw treated flies (Figure 14). Taken together these results indicate that yeast strains modified to increase levels of RNAi effectors by modulating levels of RNA stability and/or instability genes can be used successfully to elicit an insecticidal effect in D. melanogaster.

Example 7: Feeding based, biological activity in insects: Aedes aegypti

Introduction

[00167] The mosquito Aedes aegypti is a serious vector of a range of debilitating viruses, including dengue, chikungunya, and Zika virus. Currently, this mosquito is controlled by the application of broad-spectrum chemical pesticides. Through their overuse, many of these insecticides are no longer effective, as the mosquitoes have developed resistance. There are also increasing concerns about the negative impacts of these chemicals on non- target species. For these reasons, new environmentally-friendly insecticides must be developed.

[00168] A new range of species-specific pesticides is currently under development using double-stranded RNA (dsRNA). DsRNA, when it enters the cell, can induce sequence-specific knockdown of a targeted gene’s expression. Because each species has its own unique gene sequences, dsRNAs that are designed to target genes essential for a mosquito’s growth or development can potentially be used to selectively kill mosquitoes, without adversely affecting other species. This technology promises to dramatically reduce the environmental impact of mosquito larvicides. The high cost of producing dsRNA has prevented widespread adoption of RNAi, as have the challenges in stabilizing dsRNA against degradation in the aquatic environments where mosquitoes breed. To cheaply produce dsRNA stabilized within cells, yeast strains, in which high concentrations of dsRNA accumulate, were used. Yeasts are a typical food source for larval mosquitoes, and have been shown to be acceptable in dengue-endemic communities (Duman- Scheel et al. 2018).

[00169] RNAi can be used against a variety of gene targets in the mosquito, including genes specific to the brain (Hapairai et al. 2017) and cuticle (Lopez et al. 2019), and ubiquitously expressed genes (Whyard et al. 2009). In the experiments presented here, the neuronal gene fasciculation and elongation protein zeta 2 ( fez2 ) was investigated, which has been used previously in a yeast-based RNAi insecticide formulation (Hapairai et al. 2017).

Methods

Hairpin RNA construct development

[00170] A yeast RNAi system was developed containing the constitutive strong promoter ( TEF1 ), a short hairpin DNA sequence targeting Ae. aegypti fez2 (-200 bp stem sequences and 74 bp loop sequence (SEQ ID NO: 34)), a CYC1 terminator, a NatMX resistance marker cassette and TRP1 flanking regions. This cassette was assembled in the expression vector pRS423- KanMX using Gibson cloning. All the fragments required for the Gibson reaction were PCR amplified and purified, excluding the backbone vector (digested by EcoRV) and reporter gene cassette (digested by Kpnl and Sail). The assembled 2.6 kb reporter system was harvested by restriction enzyme digestion ( Bst1107Z) followed by DNA gel purification. The purified DNA fragment was used as donor DNA for integrating the reporter system into the TRP1 locus of the haploid laboratory S. cerevisiae strain BY4742.

[00171] The following strains were used in this study: BY4742, BY4742 Arrp6 Aski3, BY4742 plasmid-fez2, BY4742 Arrp6 Aski3 plasmid-fez2. Mosquito rearing and feeding

[00172] Ae. aegypti were reared under standard laboratory conditions with a 16:8 light: dark cycle and 65% humidity. Adults were fed on EDTA- treated rat blood, and eggs were collected on wet paper towels and kept moist in plastic bags. Eggs were hatched in boiled ddhhO by bubbling nitrogen for 5 minutes. Within 2 hours of hatching, 40 larvae were placed in 90 mm petri dishes with 20ml_ of ddhteO and yeast feeding pellets.

[00173] Yeast feeding pellets were prepared by inoculating 7 ml_ of YEG media with a loopful of each strain and incubating with shaking overnight at 30°C. This starter broth was used to inoculate 300ml_ of YEG media which was incubated overnight at 30°C. Cells were pelleted at 2000xg for 5 min and resuspended in 2.5ml_ of 0.7% molten agar per gram of wet mass. This suspension was heat-killed at 80°C for 20 minutes, vortexed briefly and poured into 10ml_ open-barrel syringes. After solidification, two 0.5ml_ feeding pellets were cut from the syringe using a clean cover-slip and added directly to petri dishes with mosquito larvae. For all treatments, fresh pellets were added on day 3.

[00174] Mortality was recorded on days 3 and 6 by removing mosquitoes from the water with a transfer pipette. Larvae were scored as dead if no movement was observed during processing. The developmental stage of each individual was also recorded.

Results

[00175] Compared to two negative control yeast strains lacking fez2 RNAi effector expression (BY4742 and BY4742 Arrp6 Aski3), as well as a no treatment control, increased mortality was observed in mosquitoes fed yeast strains expressing fez2 RNAi effectors (BY4742 plasmid-fez2 and BY4742 Arrp6 Aski3 plasmid-fez2). In both cases, the wildtype and Arrp6 Aski3 strains expressing fez2 RNAi effectors decreased survival of mosquitoes to approximately 50% (Figure 14). [00176] These findings indicate that yeast strains modified to increase levels of RNAi effectors by modulating levels of RNA stability and/or instability genes can be used successfully to elicit an insecticidal effect in Ae. aegypti. In this way, the modification of yeast strains does not negatively affect the functionality of the fez2 RNAi effector. It is noted however that the increased levels of fez2 RNAi effector expression observed previously in the Arrp6 Aski3 yeast (Figure 13 - 3.16 fold increase in expression in BY4742 Arrp6 Aski3 plasmid-fez2 yeast relative to BY4742 plasmid-fez2 yeast), did not translate into increased insecticidal activity. Without wishing to be bound by theory, this is likely because the choice of gene target may have a major impact on the yeast RNAi effector expression levels required to induce an insecticidal effect. Indeed, fez2 is known to be highly insecticidal in mosquitos due to its highly essential gene function (Whyard et al. 2009). Therefore, it can be expected that even modest levels of knockdown would be capable of inducing an insecticidal effect. Further experimentation using lower doses of yeast expressing fez2 RNAi effectors, or indeed yeast targeting other less-critical genes, would be expected to demonstrate an improved insecticidal effect when comparing Arrp6 Aski3 yeast and wildtype yeast.

Example 8: Feeding based, biological activity in animals: Mus musculus and Inflammatory Bowel Disease

[00177] Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, is characterized by chronic, relapsing and remitting, or progressive inflammation of the intestine. Patients with IBD suffer from intestinal inflammation and experience symptoms such as pain, nausea, and diarrhea. Crohn’s disease can occur in any part of the gastrointestinal tract, whereas ulcerative colitis is restricted to the colon. The immune response is critical in regulating host homeostasis during development of IBD and production of cytokines by immune cells contributes to intestinal inflammation. This is especially evident for production of the pro- inflammatory cytokine I L-1 b, which is the master regulator of inflammation (Coccia et al. 2012). I L-1 b plays a key role in the development of IBD by activating multiple types of immune cells. Progression of intestinal inflammation in patients with IBD is associated with increased levels of I L-1 b production (Coccia et al. 2012). To better understand the effects of different factors involved in IBD pathogenesis, murine models have been used to model intestinal inflammation. Though none of the murine models available completely represent the features of IBD in humans, they have been crucial for investigating the contribution of various factors important for pathogenesis of IBD.

A mouse model of Crohn’s disease-like intestinal inflammation

[00178] The Src homology 2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP) is a hematopoietic-specific negative regulator of the phosphatidylinositol-3-kinase (PI3K) pathway. SHIP blunts PI3K activity by removing the 5' phosphate group from class IA PI3K-generated phosphatidylinositol 3,4,5-triphosphate, an important second messenger in the cell membrane. SHIP expression levels and activity are reduced in the inflamed intestinal tissue from people with Crohn’s disease. Similar to humans, SHIP deficient mice develop spontaneous intestinal Crohn’s disease-like inflammation. Ileal inflammation is caused by increased production of macrophage-derived I L-1 b (Ngoh et al. 2016).

A mouse model mimicking ulcerative colitis

[00179] The mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1 ) is a ubiquitously expressed protein and is one of the components critical for activation of NFKB, a family of inducible transcription factors that regulates the expression of a wide variety of genes involved in immune and inflammatory responses. Maltl deficiency in humans causes dramatic inflammation along the gastrointestinal tract (McKinnon et al. 2014). Maltl deficiency (Maltl^) in mice does not result in spontaneous intestinal inflammation but it does exacerbate dextran sodium sulfate (DSS)-induced inflammation (Monajemi et al. 2018). Of note, DSS-induced colitis is a short-term, acute model of intestinal inflammation that occurs in the colon. DSS-induced colitis in Maitl^ mice is caused by increased production of I L-1 b (Monajemi et al. 2018).

Material and methods

DSS-induced colitis

[00180] Colitis was induced in Malt1- mice by adding 2% DSS to their drinking water for 6 days. Mice were monitored daily to measure disease activity index (DAI). DAI was scored on a scale of 0-12 calculated as a sum of the 0-4 score for each of the following parameters: weight loss 0-4, stool consistency 0-4, and rectal bleeding 0-4. A score of 0 = no weight loss, normal stool consistency, no rectal bleeding; 1 = 1-3% weight loss, loose stool, and detectable blood by HEMDETECT paper (Beckman Coulter, Mississauga, Canada); 2 = 3-6% weight loss, very loose stool, and visible blood in stool; 3 = 6-9% weight loss, diarrhoea, and occult blood in stool; and 4 = more than 9% weight loss, no formed stool, and extensive blood in stool and blood visible at the anus. Colons were harvested from mice and fixed in 10% formalin overnight.

Long hairpin RNA (IhRNA) treatment

[00181] Maitl^- mice were orogastrically gavaged (feeding tube) with yeast containing IhRNA constructs (SEC ID NO: 41 ) on days 0, 2, and 4 during development of DSS-induced colitis. S/-//P ^-7- were orogastrically gavaged on days 0 and 2 with a yeast concentration of 1x10 ⁹ yeast/m L and harvested on Day 10. S/-//P ^-7- were also orogastrically gavaged on days 0, 4, 8, and 12 with a yeast concentration of 2x10 ⁸ yeast/mL and harvested on Day 14. Administered fluid volumes of 5 mL/kg body weight were determined for each mouse. Control mice were gavaged with 5 mL/kg body weight of control yeast, as a vehicle control. [00182] The following strains were used in this study: BY4742 Arrp6 Aski3 empty plasmid (control), and BY4742 Arrp6 Aski3 plasmid-IL1 B-2 (test).

Histology analysis

[00183] After autopsy, tissue sections were embedded in paraffin, and cross-sections were stained with H&E. Histological damage was scored using a 16-point scale by 2 individuals blinded to the experimental conditions. Scoring included: loss of architecture 0-4; immune cell infiltration 0-4; goblet cell depletion 0-2; ulceration 0-2; edema 0-2; and muscle thickening 0-2.

Results

Yeast harbouring IhRNA decreased histological damage in SHIP deficient mice

[00184] It was asked whether blocking I L-1 b production by yeast containing IhRNA targeting I L-1 b (SEQ ID NO: 41 ) in reduced development of spontaneous ileitis in SHIP deficient mice. Development of gross inflammation in the distal ileum of SHIP deficient mice is evident by at six weeks of age (McLarren et al. 201 1 ). 6-week-old SHIP deficient mice were treated with yeast containing IhRNA for either 10 or 14 days. Ilea were fixed for histological analysis. Histological damage was scored by assessing loss of architecture, immune cell infiltration, goblet cell depletion, ulceration, edema 0-2, and muscle thickening. These facets were reduced in SHIP deficient mice treated with IhRNA compared to sham-treated mice (Figure 16A and 16B), thus resulting in a lower (better) histological damage score.

LhRNA targeting IL-Ib ameliorates DSS-induced colitis in Maltl deficient mice

[00185] It was asked whether blocking I L-1 b production by yeast containing IhRNA reduces DSS-induced intestinal inflammation in Maitl^- mice. Maitl^ mice were subjected to 2% DSS treatment to induce colitis and treated with yeast containing IhRNA (SEQ ID NO: 41 ) or control yeast. DAIs were monitored daily and after 6 days of treatment with DSS, mice were euthanized, and colons were harvested. Yeast containing IhRNA treatment decreased DAI modestly (Figure 17A). Distal colons were fixed for histological analysis. There was also modest improvement in histological damage observed in mice treated with yeast containing IhRNA (Figure 17B), as assessed by loss of architecture, immune cell infiltration, goblet cell depletion, ulceration, edema, and muscle thickening. Finally, survival rate was calculated based on humane endpoint (>15% weight loss) for Maltl^ mice treated with yeast containing IhRNA. Treatment with yeast containing IhRNA increased survival in Maltl^ mice compared to sham treated mice (Figure 17C).

[00186] While the present disclosure has been described with reference to what are presently considered to be the examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00187] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1 - Primers and sequences

SEQ ID NO:1 : RNAiEffector

5

ACT AGT GT GT GCCC AAT AGAAAG AG AACAATT G ACCCGGTT ATT GCAAGG AAAATTT CAA GT CTT GT AAAAGCAT AT AAAAAT AGTT C AGGCACT CCGAAAT ACTT GGTT GGCGT GTTT C GT AAT CAACCT AAGGAGG AT GTTTT GGCT CT GGT CAAT GATT ACGGCATT GAT AT CGT CC AACT GC AT GGAGGGT ACCCAT AGCTT CAAAAT GTTT CT ACT CCTTTTTT ACT CTT CCAG AT

1 0 TTT CT CGGACT CCGCGCAT CGCCGT ACC ACTT CAAAAC ACCC AAGC AC AGC AT ACT AAAT TT CCCCT CTTT CTT CCT CT AGGGT GT CGTT AATT ACCCGT ACT AAAGGTTT GGAAAAG AA AAAAG AG ACCGCCT CGTTT CTTTTT CTT CGT CGAAAAAGGCAAT AAAAATTTTT AT CACGT TT CTTTTT CTT G AAAATTTTTTTTTT G ATTTTTTT CT CTTT CG ATG ACCT CCC ATT GAT ATTT AAGTT AAT AAACGGT CTT CAATTT CT C AAGTTT C AGTTT C ATTTTT CTT GTT CT ATT ACAAC

15 TTTTTTT ACTT CTT GCT CATT AG AAAG AAAGC AT AGCAAT CT AAT CT AAGT CT AGAACGCT AAGT CGG AGG ACGG ACGGT CAGGT ACT AGCGGCGGT GT CT AGTTT GCT CTT GCCAT CA AC AAT GCGT GCCAT GCCTTTT CT CG AAT GT ATTTT AC AATTT CT GAAG ACGT CGGGATT G GAAAT CCCAAAGT ATT AAT AAGCACATT GTTT AT AAG ACT CGCAT GT AT GTT AAT ACT GT G GAT CCGT G AGTTT CT ATT CGCAGT CGGCT GAT CT GT GT G AAAT CTT AAT AAAGGGT CCAA TT ACCAATTT G AAACT CAGG AATT C ACAGT ATT AAC AT ACAT GCG AGT CTT AT AAACAAT G T GCTT ATT AAT ACTTT GGG ATTT CCAAT CCCG ACGT CTT CAGAAATT GT AAAAT ACATT CG AG AAAAGGC AT GGCACGC ATT GTT GAT GGCAAGAGCAAACT AG ACACCGCCGCT AGT AC CT G ACCGT CCGT CCT CCG ACTT AGCGT AAGCTTT CAT GT AATT AGTT AT GT CACGCTT AC ATT CACGCCCT CCCCCC AC AT CCGCT CT AACCG AAAAGG AAGG AGTT AGAC AACCT GAA GTCTAGGTCCCT ATTT ATTTTTTT AT AGTT ATGTT AGT ATT AAGAACGTTATTTATATTTCAA ATTTTT CTTTTTTTT CT GT ACAG ACGCGT GT ACGCAT GT AACATT AT ACT G AAAACCTT GC TT GAG AAGGTTTT GGGACGCT CGAAGGCTTT AATTT GCGT CGACGGACAT GGAGGCCCA GAAT ACCCT CCTT G ACAGT CTT GACGT GCGC AGCT CAGGGGCAT GAT GT G ACT GT CGCC CGT ACATTT AGCCCAT ACAT CCCCAT GT AT AAT CATTT GCAT CCAT ACATTTT GAT GGCCG CACGGCGCGAAGCAAAAATT ACGGCT CCT CGCT GCAG ACCT GCGAGCAGGGAAACGCT CCCCT CACAG ACGCGTT G AATT GT CCCC ACGCCGCGCCCCT GT AGAG AAAT AT AAAAGG TT AGG ATTT GCCACT G AGGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT GCT AGGAT AC AGTT CT CACAT CAC AT CCGAAC AT AAACAACC AT GGGT ACCACT CTT GACG ACACGGCTT ACCGGT ACCGCACCAGT GT CCCGGGGGACGCCG AGGCCAT CGAGGCACT GGAT GGGT CCTT C ACCACCG ACACCGT CTT CCGCGT CACCGCCACCGGGG ACGGCTT CACCCT GCG GG AGGT GCCGGT GG ACCCGCCCCT G ACCAAGGT GTT CCCCG ACGACG AAT CGGACG A CGAAT CGG ACGACGGGGAGGACGGCGACCCGGACT CCCGG ACGTT CGT CGCGT ACGG GG ACGACGGCGACCT GGCGGGCTT CGT GGT CAT CT CGT ACT CGGCGT GG AACCGCCG GCT GACCGT CG AGG ACAT CG AGGT CGCCCCGGAGCACCGGGGGCACGGGGT CGGGC GCGCGTT GAT GGGGCT CGCGACGG AGTT CGCCGGCG AGCGGGGCGCCGGGCACCT CT GGCT GG AGGT CACCAACGT CAACGCACCGGCGAT CCACGCGT ACCGGCGG AT GGGGT T CACCCT CT GCGGCCT GGACACCGCCCT GT ACG ACGGCACCGCCT CGGACGGCGAGC GGCAGGCGCT CT ACAT GAGC AT GCCCT GCCCCT AAT CAGT ACT GAC AAT AAAAAG ATT C TT GTTTT CAAGAACTT GT CATTT GT AT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT CAA AT GTT AGCGT GATTT AT ATTTTTTTT CGCCT CG ACAT CAT CT GCCCAG AT GCGAAGTT AAG T GCGCAG AAAGT AAT AT CAT GCGT CAAT CGT AT GT GAAT GCT GGT CGCT AT ACT GGT CG A CCAAG AAT ACC AAGAGTT CCT CGGTTT GCCAGTT ATT AAAAGACT CGT ATTT CCAAAAGA CT GCAACAT ACT ACT CAGT GCAGCTT CACAG AAACCT CATT CGTTT ATT CCCTT GTTT GAT T CAG AAGCAGGT GGGACAGGT GAACTTTT GG ATT GG AACT CGATTT CT GACT GGGTT GG AAGGCAAGAGGAGCT C

SEQ ID N0:2: pRS423-RNAiEffector

T CGCGCGTTT CGGT GAT G ACGGT GAAAACCT CT GACACAT GC AGCT CCCGGAG ACGGT CACAGCTT GT CT GT AAGCGGAT GCCGGGAGCAGACAAGCCCGT CAGGGCGCGT CAGC GGGT GTT GGCGGGT GT CGGGGCT GGCTT AACT AT GCGGCAT C AGAGCAG ATT GT ACT G AG AGT GC ACCAT AG ACAT GGAGGCCC AGAAT ACCCT CCTT G ACAGT CTT GACGT GCGCA GCT CAGGGGCAT GAT GT GACT GT CGCCCGT ACATTT AGCCCAT ACAT CCCCAT GT AT AA T CATTT GCAT CCAT ACATTTT GAT GGCCGCACGGCGCG AAGCAAAAATT ACGGCT CCT C GCT GCAG ACCT GCGAGCAGGGAAACGCT CCCCT CACAG ACGCGTT G AATT GT CCCCAC GCCGCGCCCCT GT AGAG AAAT AT AAAAGGTT AGG ATTT GCCACT G AGGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT GCT AGGAT ACAGTT CT CACAT CACAT CCGAAC AT AAACAAC CAT GGGT AAGG AAAAGACT CACGTTT CG AGGCCGCG ATT AAATT CCAACAT GGAT GCT G ATTT AT AT GGGT AT AAAT GGGCT CGCGAT AAT GT CGGGC AAT CAGGT GCG ACAAT CT AT C GATT GT AT GGGAAGCCCG AT GCGCCAGAGTT GTTT CT GAAACAT GGCAAAGGT AGCGTT GCCAAT GAT GTT ACAG AT G AGAT GGT CAG ACT AAACT GGCT G ACGGAATTT AT GCCT CTT CCG ACCAT CAAGC ATTTT AT CCGT ACT CCT GAT GAT GCAT GGTT ACT CACCACT GCGAT C CCCGGCAAAAC AGCATT CCAGGT ATT AGAAGAAT AT CCT GATT CAGGT G AAAAT ATT GTT GAT GCGCT GGCAGT GTT CCT GCGCCGGTT GC ATT CG ATT CCT GTTT GT AATT GT CCTTTT AAC AGCGAT CGCGT ATTT CGT CT CGCT CAGGCGCAAT CACG AAT GAAT AACGGTTT GGT T GAT GCG AGT G ATTTT GAT GACG AGCGT AAT GGCT GGCCT GTT GAACAAGT CT GGAAAG AAAT GCAT AAGCTTTT GCCATT CT CACCGGATT C AGT CGT C ACT CAT GGT G ATTT CT CAC TT GAT AACCTT ATTTTT GACGAGGGGAAATT AAT AGGTT GT ATT GAT GTTGGACG AGT CG GAAT CGCAG ACCG AT ACCAGGAT CTT GCCAT CCT AT GG AACT GCCT CGGT G AGTTTT CT CCTT C ATT ACAGAAACGGCTTTTT CAAAAAT AT GGT ATT GAT AAT CCT GAT AT G AAT AAAT T GCAGTTT CATTT GAT GCT CGAT G AGTTTTT CT AAT CAGT ACT G ACAAT AAAAAG ATT CTT GTTTT C AAG AACTT GT CATTT GT AT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT C AAAT GTT AGCGT GATTT AT ATTTTTTTT CGCCT CG ACAT CAT CT GCCCAGAT GCG AAGTT AAGT GCGCAGAAAGT AAT AT CAT GCGT CAAT CGT AT GT GAAT GCT GGT CGCT AT ACT GT AT GCG GT GT GAAAT ACCGC ACAG AT GCGT AAGGAG AAAAT ACCGCAT C AGG AAATT GT AAACGT T AAT ATTTT GTT AAAATT CGCGTT AAATTTTT GTT AAAT CAGCT CATTTTTT AACCAAT AGG CCG AAAT CGGC AAAAT CCCTT AT AAAT CAAAAGAAT AG ACCG AGAT AGGGTT GAGT GTT G TT CCAGTTT GG AACAAGAGT CCACT ATT AAAG AACGT GGACT CCAACGT CAAAGGGCG A AAAACCGT CT AT CAGGGCG AT GGCCCACT ACGT GAACCAT CACCCT AAT CAAGTTTTTT G GGGT CG AGGT GCCGT AAAGCACT AAAT CGG AACCCT AAAGGGAGCCCCCG ATTT AG AG CTT GACGGGGAAAGCCGGCG AACGT GGCGAG AAAGGAAGGG AAG AAAGCG AAAGGAG CGGGCGCT AGGGCGCT GGCAAGT GT AGCGGT CACGCT GCGCGT AACCACC ACACCCG CCGCGCTT AAT GCGCCGCT ACAGGGCGCGT CGCGCCATT CGCCATT CAGGCT GCGCAA CT GTT GGG AAGGGCG AT CGGT GCGGGCCT CTT CGCT ATT ACGCCAGCT GGCGAAAGGG GG AT GT GCT GCAAGGCG ATT AAGTT GGGT AACGCCAGGGTTTT CCC AGT CACG ACGTT G T AAAACG ACGGCC AGT GAGCGCGCGT AAT ACG ACT CACT AT AGGGCG AATTGGGT ACC GGGCCCCCCCT CG AGGT CGACGGT AT CGAT AAGCTT GAT ACT AGT GT GT GCCCAAT AG A AAG AG AACAATT G ACCCGGTT ATT GCAAGGAAAATTT CAAGT CTT GT AAAAGC AT AT AAA AAT AGTT CAGGCACT CCG AAAT ACTT GGTT GGCGT GTTT CGT AAT CAACCT AAGGAGGAT GTTTT GGCT CT GGT CAAT GATT ACGGCATT GAT AT CGT CCAACT GCAT GG AGGGT ACCC AT AGCTT CAAAAT GTTT CT ACT CCTTTTTT ACT CTT CCAGATTTT CT CGG ACT CCGCGCAT CGCCGT ACCACTT CAAAACACCC AAGC ACAGCAT ACT AAATTT CCCCT CTTT CTT CCT CT AGGGT GT CGTT AATT ACCCGT ACT AAAGGTTT GGAAAAG AAAAAAGAG ACCGCCT CGTTT CTTTTT CTT CGT CG AAAAAGGCAAT AAAAATTTTT AT CACGTTT CTTTTT CTT GAAAATTTT TTTTTT G ATTTTTTT CT CTTT CGAT G ACCT CCCATT GAT ATTT AAGTT AAT AAACGGT CTT C AATTT CT C AAGTTT CAGTTT CATTTTT CTT GTT CT ATT ACAACTTTTTTT ACTT CTT GCT CAT T AGAAAG AAAGCAT AGCAAT CT AAT CT AAGT CT AG AACGCT AAGT CGGAGGACGG ACGG T CAGGT ACT AGCGGCGGT GT CT AGTTT GCT CTT GCCAT C AAC AAT GCGT GCCAT GCCTT TT CT CG AAT GT ATTTT ACAATTT CT G AAGACGT CGGG ATT GG AAAT CCCAAAGT ATT AAT A AGCAC ATT GTTT AT AAGACT CGC AT GT AT GTT AAT ACT GT GGAT CCGT GAGTTT CT ATT CG CAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT C AGG AATT CAC AGT ATT AAC AT ACAT GCG AGT CTT AT AAACAAT GT GCTT ATT AAT ACTTT GGG A TTT CCAAT CCCG ACGT CTT CAG AAATT GT AAAAT ACATT CGAG AAAAGGC AT GGCACGC A TT GTT GAT GGC AAG AGC AAACT AGAC ACCGCCGCT AGT ACCT G ACCGT CCGT CCT CCGA CTT AGCGT AAGCTTT CAT GT AATT AGTT AT GT CACGCTT ACATT CACGCCCT CCCCCC AC AT CCGCT CT AACCGAAAAGG AAGG AGTT AG AC AACCT G AAGT CT AGGT CCCT ATTT ATTT TTTT AT AGTT AT GTT AGT ATT AAG AACGTT ATTT AT ATTT CAAATTTTT CTTTTTTTT CT GT A CAGACGCGT GT ACGCAT GT AACATT AT ACT G AAAACCTT GCTT GAG AAGGTTTT GGGAC GCT CGAAGGCTTT AATTTGCGT CG ACGGACAT GG AGGCCCAG AAT ACCCT CCTT G ACAG T CTT G ACGT GCGCAGCT C AGGGGCAT GAT GT GACT GT CGCCCGT ACATTT AGCCCAT AC AT CCCC AT GT AT AAT CATTT GCAT CCAT ACATTTT GAT GGCCGCACGGCGCGAAGC AAAA ATT ACGGCT CCT CGCT GCAG ACCT GCGAGCAGGGAAACGCT CCCCT C AC AG ACGCGTT GAATT GT CCCCACGCCGCGCCCCT GT AG AGAAAT AT AAAAGGTT AGG ATTT GCCACT G A GGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT GCT AGGAT AC AGTT CT CACAT C ACAT C CGAAC AT AAACAACC AT GGGT ACCACT CTT G ACGACACGGCTT ACCGGT ACCGCACC AG T GT CCCGGGGGACGCCGAGGCCAT CG AGGCACT GGAT GGGT CCTT CACCACCGAC AC CGT CTT CCGCGT CACCGCCACCGGGG ACGGCTT CACCCT GCGGGAGGT GCCGGT GGA CCCGCCCCT G ACCAAGGT GTT CCCCGACG ACGAAT CGGACG ACG AAT CGGACG ACGG GG AGG ACGGCG ACCCGG ACT CCCGG ACGTT CGT CGCGT ACGGGG ACGACGGCGACCT GGCGGGCTT CGT GGT CAT CT CGT ACT CGGCGT GGAACCGCCGGCT GACCGT CG AGG A CAT CG AGGT CGCCCCGG AGCACCGGGGGCACGGGGT CGGGCGCGCGTT GAT GGGGC T CGCG ACGG AGTT CGCCGGCGAGCGGGGCGCCGGGCACCT CT GGCT GGAGGT CACCA ACGT CAACGC ACCGGCG AT CCACGCGT ACCGGCGGAT GGGGTT CACCCT CT GCGGCCT GG AC ACCGCCCT GT ACGACGGCACCGCCT CGGACGGCG AGCGGCAGGCGCT CT ACAT GAGC AT GCCCT GCCCCT AAT CAGT ACT GAC AAT AAAAAG ATT CTT GTTTT CAAG AACTT G T CATTT GT AT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT CAAAT GTT AGCGT GATTT AT ATTTTTTTT CGCCT CGACAT CAT CT GCCCAGAT GCG AAGTT AAGT GCGCAGAAAGT AAT A T CAT GCGT C AAT CGT AT GT G AAT GCT GGT CGCT AT ACT GGT CG ACCAAG AAT ACCAAGA GTT CCT CGGTTT GCCAGTT ATT AAAAG ACT CGT ATTT CC AAAAGACT GCAAC AT ACT ACT C AGT GCAGCTT CAC AG AAACCT CATT CGTTT ATT CCCTT GTTT GATT CAGAAGCAGGT GGG AC AGGT G AACTTTT GG ATT GG AACT CGATTT CT GACT GGGTT GG AAGGCAAG AGG AGCT CAT CG AATT CCT GCAGCCCGGGGG AT CCACT AGTT CT AG AGCGGCCGCCACCGCGGT G GAGCT CC AGCTTTT GTT CCCTTT AGT G AGGGTT AATT GCGCGCTT GGCGT AAT CAT GGT C AT AGCT GTTT CCT GT GT G AAATT GTT AT CCGCT CACAATT CCAC AC AAC AT AGG AGCCGG AAGCAT AAAGT GT AAAGCCT GGGGT GCCT AAT G AGT GAGGT AACT CACATT AATT GCGTT GCGCT C ACT GCCCGCTTT CCAGT CGGG AAACCT GT CGT GCCAGCT GCATT AAT G AAT CG GCCAACGCGCGGGG AGAGGCGGTTT GCGT ATT GGGCGCT CTT CCGCTT CCT CGCT CAC T GACT CGCT GCGCT CGGT CGTT CGGCT GCGGCG AGCGGT AT CAGCT CACT CAAAGGCG GT AAT ACGGTT AT CC ACAGAAT C AGGGG AT AACGCAGGAAAG AAC AT GT GAGCAAAAGG CCAGC AAAAGGCC AGGAACCGT AAAAAGGCCGCGTT GCT GGCGTTTTT CC AT AGGCT CC GCCCCCCT GACG AGC AT CAC AAAAAT CGACGCT CAAGT CAGAGGT GGCG AAACCCG AC AGGACT AT AAAG AT ACC AGGCGTTT CCCCCT GG AAGCT CCCT CGT GCGCT CT CCT GTT C CGACCCT GCCGCTT ACCGG AT ACCT GT CCGCCTTT CT CCCTT CGGG AAGCGT GGCGCTT T CT CAT AGCT C ACGCT GT AGGT AT CT CAGTT CGGT GT AGGT CGTT CGCT CCAAGCT GGG CT GT GT GCACGAACCCCCCGTT CAGCCCG ACCGCT GCGCCTT AT CCGGT AACT ATCGTC TT GAGT CCAACCCGGT AAG ACACGACTT AT CGCCACT GGCAGC AGCCACT GGT AAC AGG ATT AGCAG AGCGAGGT AT GT AGGCGGT GCT ACAGAGTT CTT G AAGT GGT GGCCT AACT A CGGCT AC ACT AG AAGGACAGT ATTT GGT AT CT GCGCT CT GCT GAAGCCAGTT ACCTT CG GAAAAAG AGTT GGT AGCT CTT GAT CCGGCAAACAAACC ACCGCT GGT AGCGGT GGTTTT TTT GTTT GCAAGCAGCAG ATT ACGCGCAGAAAAAAAGG AT CT CAAGAAGAT CCTTT GAT C TTTT CT ACGGGGT CT G ACGCT CAGTGG AACGAAAACT CACGTT AAGGG ATTTT GGT CAT GAGATT AT CAAAAAGGAT CTT CACCT AG AT CCTTTT AAATT AAAAAT G AAGTTTT AAAT C AA T CT AAAGT AT AT AT GAGT AAACTT GGT CT GAC AGTT ACC AAT GCTT AAT CAGT GAGGCAC CT AT CT CAGCGAT CT GT CT ATTT CGTT CAT CCAT AGTT GCCT GACT CCCCGT CGT GT AG A T AACT ACG AT ACGGGAGGGCTT ACC AT CT GGCCCC AGT GCT GCAAT GAT ACCGCG AGAC CCACGCT CACCGGCT CCAGATTT AT CAGCAAT AAACCAGCCAGCCGGAAGGGCCGAGC GCAG AAGT GGT CCT GCAACTTT AT CCGCCT CCAT CCAGT CT ATT AATT GTT GCCGGG AA GCT AGAGT AAGT AGTT CGCCAGTT AAT AGTTT GCGCAACGTT GTT GCCATT GCT ACAGGC AT CGT GGT GT C ACGCT CGT CGTTT GGT AT GGCTT CATT CAGCT CCGGTT CCCAACGAT C AAGGCGAGTT ACAT GAT CCCCC AT GTT GT GCAAAAAAGCGGTT AGCT CCTT CGGT CCT C CGAT CGTT GT CAG AAGT AAGTT GGCCGCAGT GTT AT CACT CAT GGTT AT GGC AGCACT G CAT AATT CT CTT ACT GT CAT GCCAT CCGT AAG AT GCTTTT CT GT GACT GGT GAGT ACT C AA CCAAGT CATT CT GAG AAT AGT GT AT GCGGCG ACCG AGTT GCT CTT GCCCGGCGT CAAT A CGGGAT AAT ACCGCGCCACAT AGCAG AACTTT AAAAGT GCT CAT CATT GGAAAACGTT CT T CGGGGCG AAAACT CT CAAGG AT CTT ACCGCT GTT GAG AT CCAGTT CGAT GT AACCCAC T CGT GCACCCAACT GAT CTT CAGCAT CTTTT ACTTT CACCAGCGTTT CT GGGT GAGCAAA AAC AGG AAGGCAAAAT GCCGCAAAAAAGGGAAT AAGGGCGACACGG AAAT GTT G AAT AC T CAT ACT CTT CCTTTTT CAAT ATT ATT G AAGC ATTT AT CAGGGTT ATT GT CT CAT GAGCGG AT ACAT ATTT GAAT GT ATTT AG AAAAAT AAAC AAAT AGGGGTT CCGCGCACATTT CCCCG A AAAGT GCC ACCT G AACG AAGC AT CT GT GCTT CATTTT GT AG AACAAAAAT GCAACGCGAG AGCGCT AATTTTT CAAACAAAGAAT CT GAGCT GCATTTTT ACAGAACAGAAAT GCAACGC GAAAGCGCT ATTTT ACC AACGAAG AAT CT GT GCTT C ATTTTT GT AAAACAAAAAT GCAACG CGAG AGCGCT AATTTTT CAAACAAAGAAT CT GAGCT GCATTTTT ACAGAACAGAAAT GCA ACGCG AG AGCGCT ATTTT ACCAAC AAAGAAT CT AT ACTT CTTTTTT GTT CT ACAAAAAT GC AT CCCG AG AGCGCT ATTTTT CT AAC AAAGC AT CTT AGATT ACTTTTTTT CT CCTTT GTGCG CT CT AT AAT GC AGT CT CTT GAT AACTTTTT GCACT GT AGGT CCGTT AAGGTT AGAAG AAG GCT ACTTT GGT GT CT ATTTT CT CTT CCAT AAAAAAAGCCT G ACT CC ACTT CCCGCGTTT AC T GATT ACT AGCG AAGCT GCGGGT GC ATTTTTT CAAG AT AAAGGCAT CCCCG ATT AT ATT C T AT ACCG AT GT GG ATTGCGC AT ACTTT GT GAACAGAAAGT GAT AGCGTT GAT GATT CTT C ATT GGT CAG AAAATT AT GAACGGTTT CTT CT ATTTT GT CT CT AT AT ACT ACGT AT AGG AAA T GTTT AC ATTTT CGT ATT GTTTT CG ATT C ACT CT AT G AAT AGTT CTT ACT AC AATTTTTTT GT CT AAAGAGT AAT ACT AGAGAT AAAC AT AAAAAAT GT AG AGGT CG AGTTT AGAT GCAAGTT CAAGG AGCGAAAGGT GG AT GGGT AGGTT AT AT AGGG AT AT AGCACAG AG AT AT AT AGC A AAG AG AT ACTTTT G AGC AAT GTTT GT GG AAGCGGT ATT CGCAAT ATTTT AGT AGCT CGTT AC AGT CCGGT GCGTTTTT GGTTTTTT G AAAGT GCGT CTT CAG AGCGCTTTT GGTTTT C AA AAGCGCT CT GAAGTT CCT AT ACTTT CT AG AG AAT AGGAACTT CGGAAT AGG AACTT C AAA GCGTTT CCGAAAACG AGCGCTT CCGAAAAT GCAACGCGAGCT GCGCACAT ACAGCT C AC T GTT C ACGT CGCACCT AT AT CT GCGT GTT GCCT GT AT AT AT AT AT ACAT GAG AAG AACGG CAT AGT GCGT GTTT AT GCTT AAAT GCGT ACTT AT AT GCGT CT ATTT AT GT AGGAT G AAAGG T AGT CT AGT ACCT CCT GT GAT ATT AT CCCATT CCAT GCGGGGT AT CGT AT GCTT CCTT CA GCACT ACCCTTT AGCT GTT CT AT AT GCT GCCACT CCT C AATT GG ATT AGT CT CAT CCTT CA AT GCT AT CATTT CCTTT GAT ATT GG AT CAT CT AAG AAACCATT ATT AT CAT GACATT AACCT AT AAAAAT AGGCGT AT CACG AGGCCCTTT CGT C

SEQ ID 32: Bicoid

AGTT ATT CCGTTT GGCAGCAAAAAAT CT CCGAAT CT GAAACAAAT GGT CT GCATT GATT GAAAAT ACAATTTGCT GACT ATT CTT GGT CAAAGAAT GCGC AAAT GTTT GATT AT GT CAGACACTTT GGCAT AGCAT AGAAATT GAAAAT AT CAT AT CAAAT ATT ATT GTTT AAAT GTT CGAT CTTT AAGGGT AAT CATT GGG AT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AA AGGGT CCAATT ACCAATTT GAAACT CAGGAATT CCAAT GATT ACCCTT AA AGAT CGAACATTT AAACAAT AAT ATTT GAT AT GAT ATTTT CAATTT CTATGC T AT GCCAAAGT GT CT GACAT AAT CAAACATTT GCGCATT CTTT GACCAAG AAT AGT CAGCAAATT GT ATTTT CAAT CAATGCAGACCATTT GTTT CAGATT CGGAGATTTTTT GCT GCCAAACGGAAT AACT

SEQ ID 33: Bellwether

TT AACTT GGAGCCCGACAACGT CGGT GTT GT GGT CTT CGGT AACGAT AA GCT GAT CAAGCAGGGCGAT AT CGT CAAGCGT ACCGGT GCCAT CGT GGAT GT GCCCGT CGGT GAT GAGCT GCT GGGT CGCGT CGT CGAT GCCCT GGGA AAT GCCAT CGACGGCAAGGGT GCCAT CAACACCAAGGACCGTTT CCGT G T GGGAAT CAAGGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT G T GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CCTT GATT CCCACACGGAAACGGT CCTT GGT GTT GAT GGCACCCTTGCCGT CG AT GGCATTT CCCAGGGCAT CGACGACGCGACCCAGCAGCT CAT CACCG ACGGGCACAT CCACGATGGCACCGGT ACGCTT GACGAT AT CGCCCT GCT T GAT CAGCTT AT CGTT ACCGAAGACCACAACACCGACGTT GT CGGGCT C CAAGTTAA

SEQ ID 34: Fez2

CT CCGAAGAT GAGGCCGTT GCT AACGATTT GGAT AT GCACGCATT GATT CT GGGCGGCCTT CACACT GACAAT GAT CCGAT AAAGACAGCGGAAGAG GT CAT CAAGGAAATT GACGAT ATT AT GGACGAAAGCGCCT CCGAAGACG GCATT GTT GGT AACGAAAT CAT GGAAAAAGCCAAAGAAGTT CTT GGAT CT CCCCGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT C TT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CGGGGAGAT C CAAGAACTT CTTT GGCTTTTT CCAT GATTT CGTT ACCAACAAT GCCGT CTT CGGAGGCGCTTT CGT CCAT AAT AT CGT CAATTT CCTT GAT GACCT CTT CC GCT GT CTTT AT CGGAT CATT GT CAGT GT GAAGGCCGCCCAGAAT CAAT G CGT GCAT AT CCAAAT CGTT AGCAACGGCCT CAT CTT CGGAG

SEQ ID 35: gas8

CCT GCAGAT GCGCT GCGAGAAGCT GGT CGAAGAACGCGAT CAGCT GAA GAAT AT GTT CGAGAAGT CT AT ACT GGAGCT GCAACAGAAGT CAGGTTT GA AAAATT CCTT ATT GGAGCGAAAACT AGAAT ACAT CGAGAAGCAAACGGAA CAACGGGAAGCCATTTT AGGGGAGGT GTT AT CGCTT GCCGGAAT CGAAC CGCGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CGCGGTT CGATT CCGGCAAGCGAT AACACCT CCCCT AAAAT GGCTT CCCGTT GTT CCGTTT GCTT CT CGAT GT ATT CT AGTTTT CGCT CCAAT AAGGAATTTTT CAAACCT G ACTT CT GTT GCAGCT CCAGT AT AGACTT CT CGAACAT ATT CTT CAGCT GA T CGCGTT CTT CGACCAGCTT CT CGCAGCGCAT CT GCAGG

SEQ ID 36: gnbpal

CGAGCATTT CAGCGAT AACTTT CAT ACCT AT GGACTT GT GT GGAAGCCG GACAGCAT CGCT CT GACCGT GGAT GGATT CCAGT AT GCT ACCCT GAGGG AT CGGTT CAAGCCGT ACGGT GCGGCCAACAATTT GACCCAGGCGAATTT GT GGAAT CCGGACAAT GCCAT GT CACCGTTT GAT CGAGAGTTTT ACAT AT CGCGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CGCGAT AT GT AA AACT CT CGAT CAAACGGT GACAT GGCATT GT CCGGATT CCACAAATT CGC CT GGGT CAAATT GTT GGCCGCACCGT ACGGCTT GAACCGAT CCCT CAGG GT AGCAT ACT GGAAT CCAT CCACGGT CAGAGCGAT GCT GT CCGGCTT CC ACACAAGT CCAT AGGT AT GAAAGTT AT CGCT GAAAT GCT CG

SEQ ID 37: gnbpa3

CCCGGAAGGAGT GT ACAT GGAAGT GGACGAT GAAGT GT ACT GT CAT ATT GACCCGGAAGAAGGCTT CT ACAACGAGGT GAAAGCGACGAAACCGCAA TTT GCAAACCTTT GGAGATT GAGCGGT AAT CGAAT GGCT CCGTT CGAT AA GGAGTT CTT CATT AGTTT GGGCGT CGGT GT GGGT GGT CACT ACGACTT C CACCGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CT T AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CGGT GGAAGT C GT AGT GACCACCCACACCGACGCCCAAACT AAT GAAGAACT CCTT AT CG AACGGAGCCATT CGATT ACCGCT CAAT CT CCAAAGGTTT GCAAATT GCG GTTT CGT CGCTTT CACCT CGTT GT AGAAGCCTT CTT CCGGGT CAAT AT GA CAGT ACACTT CAT CGT CCACTT CCAT GT ACACT CCTT CCGGG SEQ ID 38: boule

AACCATT GTT GAGCGAT ATT AT CATT ATT ACACT AGT GAT CAT ATT AT AAC TT ATT AACAAACT ATTT GT AGCGT AGT GAT GAT GGAGAGAGGAGT AT CGA AGAAGAGGCAGGAGAAGCAAGT CAGAT AAAT ATT AGGAAAGT AT GCGAA AAACACGT GAAT AAAAAAAAT ACACT ACT GAT CCGAGT AACGGT AGCT GG GGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CCCAGCT ACCGTT AC T CGGAT CAGT AGT GT ATTTTTTTT ATT CACGT GTTTTT CGCAT ACTTT CCT AAT ATTT AT CT GACTT GCTT CT CCT GCCT CTT CTT CGAT ACT CCT CT CT CC AT CAT CACT ACGCT ACAAAT AGTTT GTT AAT AAGTT AT AAT AT GAT CACT A GTGT AAT AAT GAT AAT ATCGCT CAACAAT GGTT

SEQ ID 39: modsp

T GAAACT CTT ACGT GT AT CGACGGTT CTTGGGACAGTT CAGT GTTT CGAT GT GAGCCCACCT GT GGAACACCAACGCCAGAT GCT GAAGCAT ACATT AT T GGAGGT CGAAAT GCCACCAT AACGGAGGT CCCAT GGCAT ACT GGAAT A T AT CGAAAT CT GGAAACAGACACCAT CGAAGAT CTT CGAT CAGAAGATT G GCGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT A AT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CGCCAAT CTT CT G AT CGAAGAT CTT CGAT GGT GT CT GTTT CCAGATTT CGAT AT ATT CCAGT AT GCCAT GGGACCT CCGTT AT GGT GGCATTT CGACCT CCAAT AAT GT AT GCT T CAGCAT CT GGCGTT GGT GTT CCACAGGT GGGCT CACAT CGAAACACT G AACT GT CCCAAG AACCGT CGAT ACACGT AAG AGTTT CA

SEQ ID 40: II_-1 b-1

T GAACT CAACT GT GAAAT GCCACCTTTT GACAGT GAT G AGAAT GACCT GT T CTTT GAAGTT GACGGACCCCAAAAGAT GAAGGGCT GCTT CCAAACCTTT GACCT GGGCT GT CCT GAT GAGAGCAT CCAGCTT CAAAT CT CGCAGCAGC ACAT CAACAAGAGCTT CAGGCAGGCAGT AT CACT CATT GT GGCT GT GGA GAGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT A AT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CT CT CCACAGCCA CAAT GAGT GAT ACT GCCT GCCT GAAGCT CTT GTT GAT GT GCTGCT GCGA GATTT GAAGCT GGAT GCT CT CAT CAGGACAGCCCAGGT CAAAGGTTT GG AAGCAGCCCTT CAT CTTTTGGGGT CCGT CAACTT CAAAGAACAGGT CATT CT CAT CACT GT CAAAAGGT GGCATTT CACAGTT GAGTT CA

SEQ ID 41 : II_-1 b-2

GCT CCGAGAT GAACAACAAAAAAGCCT CGT GCT GT CGGACCCAT AT GAG CT GAAAGCT CT CCACCT CAAT GGACAGAAT AT CAACCAACAAGT GAT ATT CT CCAT G AGCTTT GT ACAAGG AG AACCAAGCAACG ACAAAAT ACCT GTG GCCTT GGGCCT CAAAGGAAAGAAT CT AT ACCT GT CCT GT GT AAT GAAAGA CGGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT A AT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CCGT CTTT CATT AC ACAGGACAGGT AT AGATT CTTT CCTTT GAGGCCCAAGGCCACAGGT ATTT T GT CGTT GCTT GGTT CT CCTT GT ACAAAGCT CAT GGAGAAT AT CACTT GT T GGTT GAT ATT CT GT CCATT GAGGT GGAGAGCTTT CAGCT CAT AT GGGT C CGACAGCACGAGGCTTTTTT GTT GTT CAT CT CGGAGC

SEQ ID 42: II_-1 b-3

GT CTT CCT GGGAAACAACAGT GGT CAGGACAT AATT GACTT CACCAT GG AAT CCGT GT CTT CCT AAAGT AT GGGCT GGACT GTTT CT AAT GCCTT CCCC AGGGCAT GTT AAGGAGCT CCCTTTT CGT GAAT GAGCAGACAGCT CAAT C T CCAGGGGACT CCTT AGT CCT CGGCCAAGACAGGT CGCT CAGGGT CAC AAGAGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CT T AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CT CTT GT GACC CT GAGCGACCT GT CTT GGCCGAGGACT AAGGAGT CCCCT GGAGATT GA GCT GT CT GCT CATT CACGAAAAGGGAGCT CCTT AACAT GCCCT GGGGAA GGCATT AGAAACAGT CCAGCCCAT ACTTT AGGAAGACACGGATT CCAT G GT GAAGT CAATT AT GT CCT GACCACT GTT GTTT CCCAGGAAGAC

SEQ ID 43: EGFP

CGGCCACAAGTT CAGCGT GT CCGGCGAGGGCGAGGGCGAT GCCACCT A CGGCAAGCT GACCCT GAAGTT CAT CT GCACCACCGGCAAGCT GCCCGT GCCCT GGCCCACCCT CGT GACCACCCT GACCT ACGGCGT GCAGT GCTT CAGCCGCT ACCCCGACCACAT GAAGCAGCACGACTT CTT CAAGT CCGCC AT GCCCGAAGGGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAATT ACCAATTT GAAACT CAGGAATT CCTT C GGGCAT GGCGGACTT GAAGAAGT CGT GCT GCTT CAT GT GGT CGGGGT A GCGGCT GAAGCACT GCACGCCGT AGGT CAGGGT GGT CACGAGGGT GG GCCAGGGCACGGGCAGCTT GCCGGT GGT GCAGAT GAACTT CAGGGT CA GCTT GCCGT AGGT GGCAT CGCCCT CGCCCT CGCCGGACACGCT GAACT TGTGGCCG

SEQ ID 44: pRS423-HC-RNAiEffector

T CGCGCGTTT CGGT GAT GACGGT GAAAACCT CT GACACAT GCAGCT CCC GGAGACGGT CACAGCTT GT CT GT AAGCGGAT GCCGGGAGCAGACAAGC CCGT CAGGGCGCGT CAGCGGGT GTT GGCGGGT GT CGGGGCT GGCTT A ACT AT GCGGCAT CAGAGCAGATT GT ACT GAGAGT GCACCAT AGGTT AGG ATTT GCCACT GAGGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT GCT AG GAT ACAGTT CT CACAT CACAT CCGAACAT AAACAACCAT GGGT AAGGAAA AGACT CACGTTT CGAGGCCGCGATT AAATT CCAACAT GGAT GCT GATTT A T AT GGGT AT AAAT GGGCT CGCGAT AAT GT CGGGCAAT CAGGT GCGACAA T CT AT CGATT GT AT GGGAAGCCCGAT GCGCCAGAGTT GTTT CT GAAACAT GGCAAAGGT AGCGTT GCCAAT GAT GTT ACAGAT GAGAT GGT CAGACT AA ACT GGCT GACGGAATTT AT GCCT CTT CCGACCAT CAAGCATTTT AT CCGT ACT CCT GAT GAT GCAT GGTT ACT CACCACT GCGAT CCCCGGCAAAACAG CATT CCAGGT ATT AGAAGAAT AT CCT GATT CAGGT GAAAAT ATT GTT GAT GCGCT GGCAGT GTT CCT GCGCCGGTT GCATT CGATT CCT GTTT GT AATT GT CCTTTT AACAGCGAT CGCGT ATTT CGT CT CGCT CAGGCGCAAT CACG AAT GAAT AACGGTTT GGTT GAT GCGAGT GATTTT GAT GACGAGCGT AAT G GCT GGCCT GTT GAACAAGT CT GGAAAGAAAT GCAT AAGCTTTT GCCATT C T CACCGGATT CAGT CGT CACT CAT GGT GATTT CT CACTT GAT AACCTT ATT TTT GACGAGGGGAAATT AAT AGGTT GT ATT GAT GTT GGACGAGT CGGAAT CGCAGACCGAT ACCAGGAT CTT GCCAT CCT AT GGAACT GCCT CGGT GAG TTTT CT CCTT CATT ACAGAAACGGCTTTTT CAAAAAT AT GGT ATT GAT AAT CCT GAT AT G AAT AAATT GCAGTTT CATTT GAT GCT CG AT G AGTTTTT CT AA T CAGT ACT GACAAT AAAAAGATT CTT GTTTT CAAGAACTT GT CATTT GT AT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT CAAAT GTT AGCGT GATTT A T ATTTTTTTT CGCCT CGACAT CAT CT GCCCAGAT GCGAAGTT AAGT GCGC AG AAAGT AAT AT CAT GCGT CAAT CGTATGT GAAT GCTGGTCGCTAT ACT G T AT GCGGT GT GAAAT ACCGCACAGAT GCGT AAGGAGAAAAT ACCGCAT C AGGAAATT GT AAACGTT AAT ATTTT GTT AAAATT CGCGTT AAATTTTT GTT A AAT CAGCT CATTTTTT AACCAAT AGGCCGAAAT CGGCAAAAT CCCTT AT AA AT CAAAAGAAT AGACCGAGAT AGGGTT GAGT GTT GTT CCAGTTT GGAACA AGAGT CCACT ATT AAAGAACGT GGACT CCAACGT CAAAGGGCGAAAAAC CGT CT AT CAGGGCGAT GGCCCACT ACGT GAACCAT CACCCT AAT CAAGT TTTTT GGGGT CGAGGT GCCGT AAAGCACT AAAT CGGAACCCT AAAGGGA GCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAA AGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCT AGGGCGCT GGCAAGT GT AGCGGT CACGCT GCGCGT AACCACCACACCCGCCGCGCTT AAT GCG CCGCT ACAGGGCGCGT CGCGCCATT CGCCATT CAGGCT GCGCAACT GT T GGGAAGGGCGAT CGGT GCGGGCCT CTT CGCT ATT ACGCCAGCT GGCG AAAGGGGGAT GT GCT GCAAGGCGATT AAGTT GGGT AACGCCAGGGTTTT CCCAGT CACGACGTT GT AAAACGACGGCCAGT GAGCGCGCGT AAT ACG ACT CACT AT AGGGCGAATT GGGT ACCAT AGCTT CAAAAT GTTT CT ACT CC TTTTTT ACT CTT CCAGATTTT CT CGGACT CCGCGCAT CGCCGT ACCACTT CAAAACACCCAAGCACAGCAT ACT AAATTT CCCCT CTTT CTT CCT CT AGG GT GT CGTT AATT ACCCGT ACT AAAGGTTT GGAAAAGAAAAAAGAGACCGC CT CGTTT CTTTTT CTT CGT CGAAAAAGGCAAT AAAAATTTTT AT CACGTTT CTTTTT CTT GAAAATTTTTTTTTT GATTTTTTT CT CTTT CGAT GACCT CCCA TT GAT ATTT AAGTT AAT AAACGGT CTT CAATTT CT CAAGTTT CAGTTT CATT TTT CTT GTT CT ATT ACAACTTTTTTT ACTT CTT GCT CATT AG AAAG AAAGCA T AGCAAT CT AAT CT AAGT CT AGAACGCT AAGT CGGAGGACGGACGGT CA GGT ACT AGCGGCGGT GT CT AG TTT GCT CTT GCCAT CAACAAT GCGT GCC AT GCCTTTT CT CGAAT GT ATTTT ACAATTT CT GAAGACGT CGGGATT GGA AAT CCCAAAGT ATT AAT AAGCACATT GTTT AT AAGACT CGCAT GT AT GTT A AT ACT GT GGAT CCGT GAGTTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAA T CTT AAT AAAGGGT CCAATT ACCAATTT G AAACT CAGG AATT CACAGT ATT AACAT ACAT GCGAGT CTT AT AAACAAT GT GCTT ATT AAT ACTTT GGGATTT CCAAT CCCGACGT CTT CAGAAATT GT AAAAT ACATT CGAGAAAAGGCAT G GCACGCATT GTT GAT GGCAAGAGCAAACT AGACACCGCCGCT AGT ACCT GACCGT CCGT CCT CCGACTT AGCGT AAGCTTT CAT GT AATT AGTT AT GT C ACGCTT ACATT CACGCCCT CCCCCCACAT CCGCT CT AACCGAAAAGGAA

T AT CGAATT CCT GCAGCCCGGGGGAT CCACT AGTT CT AGAGCGGCCGCC ACCGCGGT GGAGCT CCAGCTTTT GTT CCCTTT AGT GAGGGTT AATT GCG CGCTT GGCGT AAT CAT GGT CAT AGCT GTTT CCT GT GT GAAATT GTT AT CC GCT CACAATT CCACACAACAT AGGAGCCGGAAGCAT AAAGT GT AAAGCC T GGGGT GCCT AAT GAGT GAGGT AACT CACATT AATT GCGTT GCGCT CAC T GCCCGCTTT CCAGT CGGGAAACCT GT CGT GCCAGCT GCATT AAT GAAT CGGCCAACGCGCGGGGAGAGGCGGTTT GCGT ATT GGGCGCT CTT CCGC TT CCT CGCT CACT GACT CGCT GCGCT CGGT CGTT CGGCT GCGGCGAGC GGT AT CAGCT CACT CAAAGGCGGT AAT ACGGTT AT CCACAGAAT CAGGG GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG AACCGT AAAAAGGCCGCGTT GCT GGCGTTTTT CCAT AGGCT CCGCCCCC CT GACGAGCAT CACAAAAAT CGACGCT CAAGT CAGAGGT GGCGAAACCC GACAGGACT AT AAAGAT ACCAGGCGTTT CCCCCT GGAAGCT CCCT CGT G CGCT CT CCT GTT CCGACCCT GCCGCTT ACCGGAT ACCT GT CCGCCTTT C T CCCTT CGGGAAGCGT GGCGCTTT CT CAT AGCT CACGCT GT AGGT AT CT CAGTT CGGT GT AGGT CGTT CGCT CCAAGCT GGGCT GT GT GCACGAACCC CCCGTT CAGCCCGACCGCT GCGCCTT AT CCGGT AACT AT CGT CTT GAGT CCAACCCGGT AAGACACGACTT AT CGCCACT GGCAGCAGCCACT GGTAA CAGGATT AGCAGAGCGAGGT AT GT AGGCGGT GCT ACAGAGTT CTT GAAG T GGT GGCCT AACT ACGGCT ACACT AGAAGGACAGT ATTT GGT AT CT GCG CT CT GCT GAAGCCAGTT ACCTT CGGAAAAAGAGTT GGT AGCT CTT GAT CC GGCAAACAAACCACCGCT GGT AGCGGT GGTTTTTTT GTTT GCAAGCAGC AGATT ACGCGCAGAAAAAAAGGAT CT CAAGAAGAT CCTTT GAT CTTTT CT ACGGGGT CT GACGCT CAGT GGAACGAAAACT CACGTT AAGGGATTTT GG T CAT GAGATT AT CAAAAAGGAT CTT CACCT AGAT CCTTTT AAATT AAAAAT GAAGTTTT AAAT CAAT CT AAAGT AT AT AT GAGT AAACTT GGT CT GACAGTT ACCAATGCTT AAT CAGT GAGGCACCT AT CT CAGCGAT CT GT CT ATTT CGT T CAT CCAT AGTT GCCT GACT CCCCGT CGT GT AGAT AACT ACGAT ACGGG AGGGCTT ACCAT CTGGCCCCAGT GCT GCAAT GAT ACCGCGAGACCCAC GCT CACCGGCT CCAGATTT AT CAGCAAT AAACCAGCCAGCCGGAAGGGC CGAGCGCAGAAGT GGT CCT GCAACTTT AT CCGCCT CCAT CCAGT CT ATT AATT GTT GCCGGGAAGCT AGAGT AAGT AGTT CGCCAGTT AAT AGTTT GCG CAACGTT GTT GCCATT GCT ACAGGCAT CGT GGT GT CACGCT CGT CGTTT GGT AT GGCTT CATT CAGCT CCGGTT CCCAACGAT CAAGGCGAGTT ACAT GAT CCCCCAT GTT GT GCAAAAAAGCGGTT AGCT CCTT CGGT CCT CCGAT CGTT GT CAGAAGT AAGTT GGCCGCAGT GTT AT CACT CAT GGTT AT GGCA GCACT GCAT AATT CT CTT ACT GT CAT GCCAT CCGT AAGAT GCTTTT CT GT GACT GGT GAGT ACT CAACCAAGT CATT CT GAGAAT AGT GT AT GCGGCGA CCGAGTT GCT CTT GCCCGGCGT CAAT ACGGGAT AAT ACCGCGCCACAT A GCAGAACTTT AAAAGT GCT CAT CATT GGAAAACGTT CTT CGGGGCGAAAA CT CT CAAGGAT CTT ACCGCT GTT GAGAT CCAGTT CGAT GT AACCCACT CG T GCACCCAACT GAT CTT CAGCAT CTTTT ACTTT CACCAGCGTTT CT GGGT GAGCAAAAACAGGAAGGCAAAAT GCCGCAAAAAAGGGAAT AAGGGCGA CACGGAAAT GTT GAAT ACT CAT ACT CTT CCTTTTT CAAT ATT ATT GAAGCA TTT AT CAGGGTT ATT GT CT CAT GAGCGGAT ACAT ATTT GAAT GT ATTT AGA AAAAT AAACAAAT AGGGGTT CCGCGCACATTT CCCCGAAAAGT GCCACC T GAACGAAGCAT CT GT GCTT CATTTT GT AGAACAAAAAT GCAACGCGAGA GCGCT AATTTTT CAAACAAAGAAT CT GAGCT GCATTTTT ACAGAACAGAA AT GCAACGCGAAAGCGCT ATTTT ACCAACGAAGAAT CT GT GCTT CATTTT T GT AAAACAAAAAT GCAACGCGAGAGCGCT AATTTTT CAAACAAAGAAT C T GAGCT GCATTTTT ACAGAACAGAAATGCAACGCGAGAGCGCT ATTTT AC CAACAAAGAAT CT AT ACTT CTTTTTT GTT CT ACAAAAAT GCAT CCCG AG AG CGCT ATTTTT CT AACAAAGCAT CTT AGATT ACTTTTTTT CT CCTTT GT GCG CT CT AT AAT GCAGT CT CTT GAT AACTTTTT GCACT GT AGGT CCGTT AAGGT T AGAAGAAGGCT ACTTT GGT GT CT ATTTT CT CTT CCAT AAAAAAAGCCT GA CT CCACTT CCCGCGTTT ACT GATT ACT AGCGAAGCT GCGGGT GCATTTTT T CAAGAT AAAGGCAT CCCCGATT AT ATT CT AT ACCGAT GT GGATT GCGCA T ACTTT GT GAACAGAAAGT GAT AGCGTT GAT GATT CTT CATT GGT CAGAA AATT AT GAACGGTTT CTT CT ATTTT GT CT CT AT AT ACT ACGT AT AGGAAAT GTTT ACATTTT CGT ATT GTTTT CGATT CACT CT AT GAAT AGTT CTT ACT ACA ATTTTTTT GTCT AAAG AGT AAT ACT AG AGAT AAACAT AAAAAAT GT AGAGG T CGAGTTT AGAT GCAAGTT CAAGGAGCGAAAGGT GGAT GGGT AGGTT AT AT AGGGAT AT AGCACAGAGAT AT AT AGCAAAGAGAT ACTTTT GAGCAAT G TTT GT GGAAGCGGT ATT CGCAAT ATTTT AGT AGCT CGTT ACAGT CCGGT G CGTTTTT GGTTTTTT GAAAGT GCGT CTT CAGAGCGCTTTT GGTTTT CAAAA GCGCT CT GAAGTT CCT AT ACTTT CT AGAGAAT AGGAACTT CGGAAT AGGA ACTT CAAAGCGTTT CCGAAAACGAGCGCTT CCGAAAAT GCAACGCGAGC T GCGCACAT ACAGCT CACT GTT CACGT CGCACCT AT AT CT GCGT GTT GC CT GT AT AT AT AT AT ACAT GAGAAGAACGGCAT AGTGCGT GTTT AT GCTT A AAT GCGT ACTT AT AT GCGT CT ATTT AT GT AGGAT GAAAGGT AGT CT AGT A CCT CCT GT GAT ATT AT CCCATT CCAT GCGGGGT AT CGT AT GCTT CCTT CA GCACT ACCCTTT AGCT GTT CT AT AT GCT GCCACT CCT CAATT GGATT AGT CT CAT CCTT CAATGCT AT CATTT CCTTT GAT ATT GGAT CAT CT AAGAAACC ATT ATT AT CAT GACATT AACCT AT AAAAAT AGGCGT AT CACGAGGCCCTTT CGTC

SEQ ID 45: TRP1 ::RPR1-RNAi

CGT CGACGGT AT CGAT AAGCTT GAT GT GT GCCCAAT AGAAAGAGAACAA TT GACCCGGTT ATTGCAAGGAAAATTT CAAGT CTT GT AAAAGCAT AT AAAA AT AGTT CAGGCACT CCGAAAT ACTT GGTT GGCGT GTTT CGT AAT CAACCT AAGGAGGAT GTTTTGGCT CT GGT CAAT GATT ACGGCATT GAT AT CGT CCA ACT GCAT GGAGCT CGGT ACCCGAGTT AAAGAT CT GCCAATT GAACAT AA CAT GGT AGTT ACAT AT ACT AGT AAT AT GGTT CGGCACACATT AAAAGT AT A AAAACT AT CT GAATT ACGAATT ACAT AT ATT GGT CAT AAAAAT CAAT CAAT CAT CGT GT GTTTT ATATGT CT CTT AT CT AAGT AT AAG AAT AT CCAT AGTT A AT ATT CACTT ACGCT ACCTTTT AACCT GT AAT CATT GT CAACAGGAT AT GT T AACGACCCACATT GAT AAACGCT AGT ATTT CTTTTT CCT CTT CTT ATT GG CCGGCT GT CT CT AT ACT CCCCT AT AGT CT GTTT CTTTT CGTTT CGATT GTT TT ACGTTT GAGGCCT CGT GGCGCACAT GGT ACGCT GT GGT GCT CGCGG CT GGGAACGAAACT CT GGGAGCT GCGATT GGCAGCAAT CT AAT CT AAGT CT AGAACGCT AAGT CGGAGGACGGACGGT CAGGT ACT AGCGGCGGT GT ATTTT ACAATTT CT GAAGACGT CGGGATT GGAAAT CCCAAAGT ATT AAT AA GCACATT GTTT AT AAGACT CGCAT GT AT GTT AAT ACT GT GGAT CCGT GAG TTT CT ATT CGCAGT CGGCT GAT CT GT GT GAAAT CTT AAT AAAGGGT CCAA TT ACCAATTT GAAACT CAGGAATT CACAGT ATT AACAT ACAT GCGAGT CTT AT AAACAAT GT GCTT ATT AAT ACTTT GGGATTT CCAAT CCCGACGT CTT CA GAAATT GT AAAAT ACATT CGAGAAAAGGCAT GGCACGCATT GTT GAT GGC AAGAGCAAACT AGACACCGCCGCT AGT ACCT GACCGT CCGT CCT CCGAC TT AGCGT AAGCTTT CAT GT CCAT AT CCAACTT CCAATTT AAT CTTT CTTTTT T AATTTT CACTT ATTTGCGAT ACAGAAAGAGGGGAT CCGACAT GGAGGCC CAGAAT ACCCT CCTT GACAGT CTT GACGTGCGCAGCT CAGGGGCAT GAT GT GACT GT CGCCCGT ACATTT AGCCCAT ACAT CCCCAT GT AT AAT CATTT GCAT CCAT ACATTTT GAT GGCCGCACGGCGCGAAGCAAAAATT ACGGCT CCT CGCT GCAGACCT GCGAGCAGGGAAACGCT CCCCT CACAGACGCGT T GAATT GT CCCCACGCCGCGCCCCT GT AGAGAAAT AT AAAAGGTT AGGA TTT GCCACT GAGGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT GCT AGG AT ACAGTT CT CACAT CACAT CCGAACAT AAACAACCAT GGGT ACCACT CT T GACGACACGGCTT ACCGGT ACCGCACCAGT GT CCCGGGGGACGCCGA GGCCAT CGAGGCACT GGAT GGGT CCTT CACCACCGACACCGT CTT CCG CGT CACCGCCACCGGGGACGGCTT CACCCT GCGGGAGGT GCCGGT GG ACCCGCCCCT GACCAAGGT GTT CCCCGACGACGAAT CGGACGACGAAT CGGACGACGGGGAGGACGGCGACCCGGACT CCCGGACGTT CGT CGCG T ACGGGGACGACGGCGACCT GGCGGGCTT CGT GGT CAT CT CGT ACT CG GCGT GGAACCGCCGGCT GACCGT CGAGGACAT CGAGGT CGCCCCGGA GCACCGGGGGCACGGGGT CGGGCGCGCGTT GAT GGGGCT CGCGACGG AGTT CGCCGGCGAGCGGGGCGCCGGGCACCT CTGGCT GGAGGT CACC AACGT CAACGCACCGGCGAT CCACGCGT ACCGGCGGAT GGGGTT CACC CT CT GCGGCCT GGACACCGCCCT GT ACGACGGCACCGCCT CGGACGGC GAGCGGCAGGCGCT CT ACAT GAGCAT GCCCT GCCCCT AAT CAGT ACT GA CAAT AAAAAG ATT CTT GTTTT CAAGAACTT GT CATTT GTAT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT CAAAT GTT AGCGT GATTT AT ATTTTTTTT C GCCT CGACAT CAT CT GCCCAGAT GCGAAGTT AAGT GCGCAGAAAGT AAT AT CAT GCGT CAAT CGT AT GT GAAT GCT GGT CGCT AT ACT GCAAGAAT ACC AAGAGTT CCT CGGTTT GCCAGTT ATT AAAAGACT CGT ATTT CCAAAAGAC T GCAACAT ACT ACT CAGT GCAGCTT CACAGAAACCT CATT CGTTT ATT CC CTT GTTT GATT CAGAAGCAGGT GGGACAGGT GAACTTTT GGATT GGAACT CGATTT CT GACT GGGTT GGAAGGCAAGAG

SEQ ID 46: pRS343-Psnr33TUB

T CGCGCGTTT CGGT GAT GACGGT GAAAACCT CT GACACAT GCAGCT CCC GGAGACGGT CACAGCTT GT CT GT AAGCGGAT GCCGGGAGCAGACAAGC CCGT CAGGGCGCGT CAGCGGGT GTT GGCGGGT GT CGGGGCT GGCTT A ACT AT GCGGCAT CAGAGCAGATT GT ACT GAGAGT GCACCAT AGACATGG AGGCCCAGAAT ACCCT CCTT GACAGT CTT GACGTGCGCAGCT CAGGGG CAT GAT GT GACT GT CGCCCGT ACATTT AGCCCAT ACAT CCCCAT GT AT AA T CATTT GCAT CCAT ACATTTT GAT GGCCGCACGGCGCGAAGCAAAAATT A CGGCT CCT CGCT GCAGACCT GCGAGCAGGGAAACGCT CCCCT CACAGA CGCGTT GAATT GT CCCCACGCCGCGCCCCT GT AGAGAAAT AT AAAAGGT T AGGATTT GCCACT GAGGTT CTT CTTT CAT AT ACTT CCTTTT AAAAT CTT G CT AGGAT ACAGTT CT CACAT CACAT CCGAACAT AAACAACCAT GGGT AAG GAAAAGACT CACGTTT CGAGGCCGCGATT AAATT CCAACAT GGAT GCTG ATTT AT ATGGGT AT AAAT GGGCT CGCGAT AAT GT CGGGCAAT CAGGT GC GACAAT CT AT CGATT GT AT GGGAAGCCCGAT GCGCCAGAGTT GTTT CT G AAACAT GGCAAAGGT AGCGTT GCCAAT GAT GTT ACAGAT GAGAT GGT CA GACT AAACT GGCT GACGGAATTT AT GCCT CTT CCGACCAT CAAGCATTTT AT CCGT ACT CCT GAT GAT GCAT GGTT ACT CACCACT GCGAT CCCCGGCA AAACAGCATT CCAGGT ATT AGAAGAAT AT CCT GATT CAGGT GAAAAT ATT GTT GATGCGCTGGCAGT GTT CCT GCGCCGGTT GCATT CGATT CCT GTTT GT AATT GT CCTTTT AACAGCGAT CGCGT ATTT CGT CT CGCT CAGGCGCAA T CACGAAT GAAT AACGGTTT GGTT GAT GCGAGT GATTTT GAT GACGAGC GT AAT GGCT GGCCT GTT GAACAAGT CT GGAAAGAAAT GCAT AAGCTTTT G CCATT CT CACCGGATT CAGT CGT CACT CAT GGT GATTT CT CACTT GAT AA CCTT ATTTTT GACGAGGGGAAATT AAT AGGTT GT ATT GAT GTT GGACGAG T CGGAAT CGCAGACCGAT ACCAGGAT CTT GCCAT CCT AT GGAACT GCCT CGGT GAGTTTT CT CCTT CATT ACAGAAACGGCTTTTT CAAAAAT AT GGT AT T GAT AAT CCT GAT AT GAAT AAATT GCAGTTT CATTT GAT GCT CG AT GAGTT TTT CT AAT CAGT ACT GACAAT AAAAAG ATT CTT GTTTT CAAG AACTT GT CA TTT GT AT AGTTTTTTT AT ATT GT AGTT GTT CT ATTTT AAT CAAAT GTT AGCG T GATTT AT ATTTTTTTT CGCCT CGACAT CAT CT GCCCAGAT GCGAAGTT AA GT GCGCAGAAAGT AAT AT CAT GCGT CAAT CGT AT GT GAAT GCTGGT CGC T AT ACT GT AT GCGGT GT GAAAT ACCGCACAGAT GCGT AAGGAGAAAAT A CCGCAT CAGGAAATT GT AAACGTT AAT ATTTT GTT AAAATT CGCGTT AAAT TTTT GTT AAAT CAGCT CATTTTTT AACCAAT AGGCCGAAAT CGGCAAAAT C CCTT AT AAAT CAAAAGAAT AGACCGAGAT AGGGTT GAGT GTT GTT CCAGT TT GGAACAAGAGT CCACT ATT AAAGAACGT GGACT CCAACGT CAAAGGG CGAAAAACCGT CT AT CAGGGCGAT GGCCCACT ACGT GAACCAT CACCCT AAT CAAGTTTTTT GGGGT CGAGGT GCCGT AAAGCACT AAAT CGGAACCC T AAAGGGAGCCCCCGATTT AGAGCTT GACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGC T GGCAAGT GT AGCGGT CACGCT GCGCGT AACCACCACACCCGCCGCGC TT AAT GCGCCGCT ACAGGGCGCGT CGCGCCATT CGCCATT CAGGCTGC GCAACT GTT GGGAAGGGCGAT CGGT GCGGGCCT CTT CGCT ATT ACGCC AGCT GGCGAAAGGGGGAT GT GCT GCAAGGCGATT AAGTT GGGT AACGC CAGGGTTTT CCCAGT CACGACGTT GT AAAACGACGGCCAGT GAGCGCG CGT AAT ACGACT CACT AT AGGGCGAATTGGGT ACCGGGCCcccggttcgattcc gggcttgcgcatcttttttactttatatactattttttttttttttctttttcccaaatt ttttcatgaaaaatttggcggaacg gtacataagaatagaagagattcgttatgaaaattttctactctctttcacatttttttt ttcataagaattaaaaaa attCT AGAACGCT AAGT CGGAGGACGGACGGT CAGGT ACT AGCGGCGGT

TT AT AAACAAT GT GCTT ATT AAT ACTTT GGGATTT CCAAT CCCGACGT CTT CAGAAATT GT AAAAT ACATT CGAGAAAAGGCAT GGCACGCATT GTT GAT G GCAAGAGCAAACT AGACACCGCCGCT AGT ACCT GACCGT CCGT CCT CCG ACTT AGCGT AAGCTTT CAT GT AATT AGTT AT GT CACGCTT ACATT CACGCC CT CCCCCCACAT CCGCT CT AACCGAAAAGGAAGGAGTT AGACAACCT GA AGT CT AGGT CCCT ATTT ATTTTTTT AT AGTT AT GTT AGT ATT AAGAACGTT A TTT AT ATTT CAAATTTTT CTTTTTTTT CT GT ACAGACGCGT GT ACGCAT GT A ACATT AT ACT GAAAACCTTGCTT GAGAAGGTTTT GGGACGCT CGAAGGCT TT AATTT GCGT CGACGGT AT CGAT AAGCTT GAT AT CGAATT CCTGCAGCC CGGGGGAT CCACT AGTT CT AGAGCGGCCGCCACCGCGGT GGAGCT CCA GCTTTT GTT CCCTTT AGT GAGGGTT AATT GCGCGCTT GGCGT AAT CAT GG T CAT AGCT GTTT CCT GT GT GAAATT GTT AT CCGCT CACAATT CCACACAA CAT AGGAGCCGGAAGCAT AAAGT GT AAAGCCT GGGGT GCCT AAT GAGT G AGGT AACT CACATT AATT GCGTT GCGCT CACTGCCCGCTTT CCAGT CGG GAAACCT GT CGT GCCAGCT GCATT AAT GAAT CGGCCAACGCGCGGGGA GAGGCGGTTT GCGT ATT GGGCGCT CTT CCGCTT CCT CGCT CACT GACT C GCT GCGCT CGGT CGTT CGGCT GCGGCGAGCGGT AT CAGCT CACT CAAA GGCGGT AAT ACGGTT AT CCACAGAAT CAGGGGAT AACGCAGGAAAGAAC AT GT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT AAAAAGGCCGCG TT GCT GGCGTTTTT CCAT AGGCT CCGCCCCCCT GACGAGCAT CACAAAA AT CGACGCT CAAGT CAGAGGT GGCGAAACCCGACAGGACT AT AAAGAT A CCAGGCGTTT CCCCCT GGAAGCT CCCT CGT GCGCT CT CCT GTT CCGACC CT GCCGCTT ACCGGAT ACCT GT CCGCCTTT CT CCCTT CGGGAAGCGT GG CGCTTT CT CAT AGCT CACGCT GT AGGT AT CT CAGTT CGGT GT AGGT CGTT CGCT CCAAGCT GGGCT GT GT GCACGAACCCCCCGTT CAGCCCGACCGC T GCGCCTT AT CCGGT AACT AT CGT CTT GAGT CCAACCCGGT AAGACACG ACTT AT CGCCACT GGCAGCAGCCACT GGT AACAGGATT AGCAGAGCGAG GT AT GT AGGCGGT GCT ACAGAGTT CTT GAAGT GGT GGCCT AACT ACGGC T ACACT AGAAGGACAGT ATTT GGT AT CT GCGCT CT GCT GAAGCCAGTT AC CTT CGGAAAAAGAGTT GGT AGCT CTT GAT CCGGCAAACAAACCACCGCT GGT AGCGGT GGTTTTTTT GTTT GCAAGCAGCAGATT ACGCGCAGAAAAA AAGGAT CT CAAGAAGAT CCTTT GAT CTTTT CT ACGGGGT CT GACGCT CAG T GGAACGAAAACT CACGTT AAGGGATTTT GGT CAT GAGATT AT CAAAAAG GAT CTT CACCT AGAT CCTTTT AAATT AAAAAT GAAGTTTT AAAT CAAT CT AA AGT AT AT AT GAGT AAACTT GGT CT G ACAGTT ACCAAT GCTT AAT CAGT GA GGCACCT AT CT CAGCGAT CT GT CT ATTT CGTT CAT CCAT AGTT GCCT GAC T CCCCGT CGT GT AGAT AACT ACGAT ACGGGAGGGCTT ACCAT CT GGCCC CAGT GCT GCAAT GAT ACCGCGAGACCCACGCT CACCGGCT CCAGATTT A T CAGCAAT AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT GGT CCT GCAACTTT AT CCGCCT CCAT CCAGT CT ATT AATT GTT GCCGGGAAGCT AG AGT AAGT AGTT CGCCAGTT AAT AGTTT GCGCAACGTT GTT GCCATT GCTA CAGGCAT CGT GGT GT CACGCT CGT CGTTT GGT AT GGCTT CATT CAGCT C CGGTT CCCAACGAT CAAGGCGAGTT ACAT GAT CCCCCAT GTT GT GCAAA GT CATT CT GAGAAT AGT GT AT GCGGCGACCGAGTT GCT CTT GCCCGGCG T CAAT ACGGGAT AAT ACCGCGCCACAT AGCAGAACTTT AAAAGT GCT CAT CATT GGAAAACGTT CTT CGGGGCGAAAACT CT CAAGGAT CTT ACCGCT G TT GAGAT CCAGTT CGAT GT AACCCACT CGT GCACCCAACT GAT CTT CAGC AT CTTTT ACTTT CACCAGCGTTT CT GGGT GAGCAAAAACAGGAAGGCAAA AT GCCGCAAAAAAGGGAAT AAGGGCGACACGGAAAT GTT GAAT ACT CAT ACT CTT CCTTTTT CAAT ATT ATT GAAGCATTT AT CAGGGTT ATT GT CT CAT GAGCGGAT ACAT ATTT GAAT GT ATTT AGAAAAAT AAACAAAT AGGGGTT C CGCGCACATTT CCCCGAAAAGT GCCACCT GAACGAAGCAT CT GT GCTT C ATTTT GT AGAACAAAAAT GCAACGCGAGAGCGCT AATTTTT CAAACAAAG AAT CT GAGCT GCATTTTT ACAGAACAGAAAT GCAACGCGAAAGCGCT ATT TT ACCAACGAAGAAT CT GT GCTT CATTTTT GT AAAACAAAAAT GCAACGC GAGAGCGCT AATTTTT CAAACAAAGAAT CT GAGCT GCATTTTT ACAGAAC AGAAATGCAACGCGAGAGCGCT ATTTT ACCAACAAAGAAT CT AT ACTT CT TTTTT GTT CT ACAAAAAT GCAT CCCGAGAGCGCT ATTTTT CT AACAAAGCA T CTT AGATT ACTTTTTTT CT CCTTT GT GCGCT CT AT AAT GCAGT CT CTT GA T AACTTTTT GCACT GT AGGT CCGTT AAGGTT AGAAGAAGGCT ACTTT GGT GT CT ATTTT CT CTT CCAT AAAAAAAGCCT G ACT CCACTT CCCGCGTTT ACT GATT ACT AGCGAAGCT GCGGGT GCATTTTTT CAAGAT AAAGGCAT CCCC GATT AT ATT CT AT ACCGAT GTGGATT GCGCAT ACTTT GT GAACAGAAAGT GAT AGCGTT GAT GATT CTT CATT GGT CAGAAAATT AT GAACGGTTT CTT CT ATTTT GT CT CT AT AT ACT ACGT AT AGGAAAT GTTT ACATTTT CGT ATT GTTT T CGATT CACT CT AT GAAT AGTT CTT ACT ACAATTTTTTT GT CT AAAG AGT AA T ACT AGAGAT AAACAT AAAAAAT GT AGAGGT CGAGTTT AGAT GCAAGTT C AAGGAGCGAAAGGT GGAT GGGT AGGTT AT AT AGGGAT AT AGCACAGAGA T AT AT AGCAAAGAGAT ACTTTT GAGCAAT GTTT GT GGAAGCGGT ATT CGC AAT ATTTT AGT AGCT CGTT ACAGT CCGGT GCGTTTTT GGTTTTTT GAAAGT GCGT CTT CAGAGCGCTTTT GGTTTT CAAAAGCGCT CT GAAGTT CCT AT AC TTT CT AGAGAAT AGGAACTT CGGAAT AGGAACTT CAAAGCGTTT CCGAAA ACGAGCGCTT CCGAAAAT GCAACGCGAGCT GCGCACAT ACAGCT CACT G TT CACGT CGCACCT ATATCTGCGT GTT GCCTGT AT AT AT AT AT ACAT GAG AAGAACGGCAT AGTGCGT GTTT AT GCTT AAAT GCGT ACTT AT AT GCGT CT ATTT AT GT AGGAT GAAAGGT AGT CT AGT ACCT CCT GT GAT ATT AT CCCATT CCAT GCGGGGT AT CGT AT GCTT CCTT CAGCACT ACCCTTT AGCT GTT CT A T AT GCT GCCACT CCT CAATT GGATT AGT CT CAT CCTT CAAT GCT AT CATTT CCTTT GAT ATT GGAT CAT CT AAGAAACCATT ATT AT CAT GACATT AACCT A T AAAAAT AGGCGT AT CACGAGGCCCTTT CGT C References

Anderson, K. E., Sheehan, T. H., Eckholm, B. J., & Mott, B. M. (201 1 ). An emerging paradigm of colony health: microbial balance of the honey bee and hive (Apis mellifera). Insectes Sociaux 58:431.

Atwood, D., & Paisley, C. (2017). Pesticides industry sales and usage 2008- 2012 market estimates. United States Environmental Protection Agency. https://www.epa.gov/sites/production/files/2017-01/documents /pesticides- industry-sales-usage-2016_0.pdf

Beketov, M. A., & Kefford, B. J. (2013). Pesticides reduce regional biodiversity of stream invertebrates. Proceedings of the National Academy of Sciences , 110(27), 1 1039. http://doi.org/10.1073/pnas.13056181 10.

Bradford, B. J., Cooper, C. A., Tizard, M. L, & Doran, T. J. (2017). RNA interference-based technology: what role in animal agriculture? Animal Production Science, 57(1 ), 1 . http://doi.org/10.1071/an15437.

Butler, D. (2010). Food: the growing problem. Nature. 466: 546-547.

Chang, Q., Wang, W., Regev-Yochay, G., Lipsitch, M., & Hanage, W. P. (2014). Antibiotics in agriculture and the risk to human health: how worried should we be? Evolutionary Applications, 8(3), 240-247. http://doi.Org/10.1 1 1 1/eva.12185

Coccia, M., et al., IL-1beta mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(+) Th17 cells. J Exp Med, 2012. 209(9): p. 1595-609.

Connolly, B., Isaacs, C., Cheng, L., Asrani, K. H., & Subramanian, R. R. (2018). SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency. Journal of nucleic acids, 2018.

DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR- Cas systems. Nucleic acids research, 41(7), 4336-4343.

Drummond, R. O., Lambert, G., Smalley Jr, H. E., & Terrill, C. E. (1981 ). Estimated losses of livestock to pests [USA] CRC Handbook of Pest Management in Agriculture (USA).

Duman-Scheel, M., Eggleson, K. K., Achee, N. L., Grieco, J. P. & Hapairai, L. K. Mosquito control practices and perceptions: An analysis of economic stakeholders during the Zika epidemic in Belize, Central America. PloS One 13, e0201075 (2018).

Fire, A., Kostas, S., Montgomery, M., Timmons, L, Xu, S., Tabara, H., ... & Mello, C. C. (2003). U.S. Patent No. 6,506,559. Washington, DC: U.S. Patent and Trademark Office.

Fujita, T., Ikuta, J., Hamada, J., Okajima, T., Tatematsu, K., Tanizawa, K., & Kuroda, S. I. (2004). Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1. Biochemical and biophysical research communications , 313(3), 738-744.

Garcia, J. F., Carbone, M. A., Mackay, T. F., & Anholt, R. R. (2017). Regulation of Drosophila Lifespan by bellwether Promoter Alleles. Scientific reports, 7(1 ), 4109.

Ghosh, S., Hunter, W. B., & Park, A. L. (2017). Double strand RNA delivery system for plant-sap-feeding insects. PLOS ONE, 12(2), e0171861. http://doi.org/10.1371/journal.pone.0171861 .

Gietz, R. D., & Schiestl, R. H. (2007). High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature protocols, 2(1 ), 31.

Giraldo-Calderon, Gloria I., et al. "VectorBase: an updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases." Nucleic acids research 43. D1 (2014): D707-D713.

Hapairai, L. K. et al. Lure-and-Kill Yeast Interfering RNA Larvicides Targeting Neural Genes in the Human Disease Vector Mosquito Aedes aegypti. Sci. Rep. 7, 13223 (2017).

Jacobs, H., R. Stratmann, and C. F. Lehner. "A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster." Molecular and General Genetics MGG 259.4 (1998): 383-387.

Jin, S., Singh, N. D., Li, L., Zhang, X., & Daniell, H. (2015). Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation. Plant biotechnology journal, 13(3), 435-446.

Joga, M. R., Zotti, M. J., Smagghe, G., & Christiaens, O. (2016). RNAi efficiency, systemic properties, and novel delivery methods for pest insect control: what we know so far. Frontiers in physiology, 1, 553. Kohrer, K., & Domdey, H. (1991 ). [27] Preparation of high molecular weight RNA. In Methods in enzymology {V ol. 194, pp. 398-405). Academic Press.

Laroui H., Theiss AL, Yan Y., Dalmasso G., Nguyen HTT., Sitaraman, SV., & Merlin, D. (201 1 ). Functional TNFa gene silencing mediated by polyethyleneimine/TNFa siRNA nanocomplexes in inflamed colon. Biomaterials 32(4): 1218-1228.

Li, X., Zhang, M., & Zhang, H. (201 1 ). RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs. PLOS ONE, 6(3), e17788. http://doi.org/10.1371/journal.pone.0017788.

Lin, Y. H., Huang, J. H., Liu, Y., Belles, X., & Lee, H. J. (2017). Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response. Pest management science, 73(5), 960-966.

Lopez, S. B. G. et al. RNAi-based bioinsecticide for Aedes mosquito control. Sci. Rep. 9, 4038 (2019).

Lu, H. L., Vinson, S. B., & Pietrantonio, P. V. (2009). Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virgin queens. The FEBS journal, 276( 1 1 ), 31 10-3123.

McKinnon, M.L., et al., Combined immunodeficiency associated with homozygous MALT1 mutations. J Allergy Clin Immunol, 2014. 133(5): p. 1458-62, 1462 e 1 -7.

McLarren, K.W., et al., SHIP-deficient mice develop spontaneous intestinal inflammation and arginase-dependent fibrosis. Am J Pathol, 201 1. 179(1 ): p. 180-8.

Monajemi, M., et al., Maltl blocks IL-1beta production by macrophages in vitro and limits dextran sodium sulfate-induced intestinal inflammation in vivo. J Leukoc Biol, 2018. 104(3): p. 557-572.

Murphy, K.A. et al. (2016) Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci Rep-uk 6, 1 13

Mysore, Keshava, et al. "Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality." Malaria journal 16.1 (2017): 461.

Mysore, Keshava, et al. "Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae." Insect Genomics. Humana Press, New York, NY, 2019. 213-231 . Ngoh, E.N., et al., Activity of SHIP, Which Prevents Expression of Interleukin 1beta, Is Reduced in Patients With Crohn's Disease. Gastroenterology, 2016. 150(2): p. 465-76.

Oerke, E. C. (2006). Crop losses to pests. The Journal of Agricultural Science , 144(1 ), 31-43.

Pagel, S. W., & Gautier, P. (2012). Use of antimicrobial agents in livestock. Revue Scientifique Et Technique (International Office of Epizootics), 31(1 ), 145-188.

Pimentel, D., & Burgess, M. (2014). Environmental and economic costs of the application of pesticides primarily in the United States. In Integrated pest management (pp. 47-71 ). Springer, Dordrecht.

Pimentel, David, and Michael Burgess. "Small amounts of pesticides reaching target insects." (2012): 1 -2.

Prieve, M. G., Harvie, P., Monahan, S. D., Roy, D., Li, A. G., Blevins, T. L., & Ella-Menye, J. R. (2018). Targeted mRNA therapy for ornithine transcarbamylase deficiency. Molecular Therapy, 26(3),801-813.

Roseman, Daniel S., et al. "G6PC mRNA therapy positively regulates fasting blood glucose and decreases liver abnormalities in a mouse model of glycogen storage disease 1 a." Molecular Therapy 26.3 (2018): 814-821.

Tiemann, K. & Rossi JJ. (2009). RNAi-based therapeutics-current status, challenges and prospects. EMBO Molecular Medicine 1 :142-151 .

Trepotec, Z., Lichtenegger, E., Plank, C., Aneja, M. K., & Rudolph, C. (2018). Delivery of mRNA therapeutics for treatment of hepatic diseases. Molecular Therapy. 27(4): 794-802.

Van Boeckel, T. P., Brower, C., & Gilbert, M. (2015). Global trends in antimicrobial use in food animals. Proceedings of the National Academy of Sciences, 112(18), 5649. http://doi.org/10.1073/pnas.1503141 112

Whitten, M., Facey, P. D., & Del Sol, R. (2016). Symbiont-mediated RNA interference in insects. Proceedings of the Royal Society B: Biological Sciences, 283(1825), 20160042. http://doi.org/10.1098/rspb.2016.0042

Whyard, S., Singh, A. D. & Wong, S. Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochem. Mol. Biol. 39, 824-832 (2009). Winzeler, Elizabeth A., et al. "Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis." science 285.5429 (1999): 901 -906.

Xiong, Q., Lee, G. Y., Ding, J., Li, W., & Shi, J. (2018). Biomedical applications of mRNA nanomedicine. Nano research , 77(10), 5281 -5309.

Yu, Xiu-Dao, et al. "RNAi-mediated plant protection against aphids." Pest management science 72.6 (2016): 1090-1098.