Genetically engineered marine bacteria for biomaterial production, use in aquaculture and marine environmental restoration and methods for making and using them

Abstract

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for non-coding RNA (such as RNAi or dsRNA) treatment to target animals or plants of interest by delivering non-coding RNA (such as RNAi or dsRNA) using genetically engineered bacteria, such as environmental and / or microbiome bacteria, and this genetic engineering allows the expression and delivery of bacterial-derived non-coding RNA (such as RNAi or dsRNA) machinery for the genetic manipulation of the target animals or plants of interest.

Description

[0001]PATENT 5810.157405PCT / Shikuma-N6 GENETICALLY ENGINEERED MARINE BACTERIA FOR BIOMATERIAL PRODUCTION, USE IN AQUACULTURE AND MARINE ENVIRONMENTAL RESTORATION AND METHODS FOR MAKING AND USING THEM RELATED APPLICATIONS This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. (USSN) 63 / 687,110, filed August 26, 2024. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with government support under 1R35GM146722 awarded by the National Institutes of Health, and 1942251 awarded by the National Science Foundation. The government has certain rights in the invention. TECHNICAL FIELD This invention generally relates to molecular and marine biology. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for transferring nucleic acid from a bacteria to a target cell, for example, transferring a non-coding RNA such as RNAi or dsRNA, dsRNA, siRNA, shRNA, antisense oligonucleotides, ribozymes, aptamers, piRNAs, lncRNAs, or DNA fragments to a target cell; and in alternative embodiments this transfer targets animals or plants of interest by delivering the nucleic acid (for example, the non-coding RNA such as RNAi or dsRNA) using genetically engineered bacteria, wherein optionally environmental and / or microbiome bacteria are genetically engineered, and this genetic engineering allows the expression and delivery of bacterial-generated nucleic acid (such as non-coding RNA such as RNAi or dsRNA) machinery for the genetic manipulation of the target animals or plants of interest. In alternative embodiments, this nucleic acid transfer to a target cell results in change of phenotype of the target cell, and optionally the change in phenotype results in treatment of a disease or condition in the target cell. PATENT 5810.157405PCT / Shikuma-N6 BACKGROUND Life-history transitions in animals are significant developmental or behavioral shifts that occur throughout an organisms’ life. For example, the emergence of an individual from an egg, the transition of a tadpole undergoing metamorphosis to a frog, or sexual maturation such as puberty in humans (Gilbert et al., 2015; Schmidt et al., 2012; Warkentin, 2011). Although microbes have been linked to cellular and tissue development of many animals, including humans (Hill et al., 2022; Mishra et al., 2021; Ruby & Asato, 1993; Sommer & Bäckhed, 2013), examples of how microbes play critical roles in major life-history transitions remain scarce. Since the 1930s, many marine animals have been known to undergo the major life-history transition of metamorphosis in response to bacteria (Zobell & Allen, 1935). During this host-microbe interaction, swimming animal larvae sense and respond to surface-bound bacteria by undergoing metamorphosis to the juvenile form and transition from a planktonic to benthic lifestyle. Animals as diverse as sponges, corals, tubeworms and urchins have been shown to undergo metamorphosis in response to bacteria (Cavalcanti et al., 2020; Hadfield, 2011), suggesting that the linkages between metamorphic development and bacterial sensing are ancient and conserved. While genes implicated in immune activation have been observed during metamorphosis across diverse phyla (Balseiro et al., 2013a; Critchlow et al., 2019; Davidson & Swalla, 2002; Natori et al., 1999), the necessity of the immune system for mediating animal metamorphosis in response to bacteria has not been demonstrated for any animal. Toll Like Receptors (TLRs) and Myeloid Differentiation Factor 88 (MyD88) are parts of an innate immune pathway that play important roles during the development of many animals. For example, nine different TLRs are utilized for either embryonic development, dorsal ventral symmetry, heart and muscle formation or bacterial defense in Drosophila melanogaster (Valanne et al., 2011), and TLR / MyD88 signaling is required for pathogen defense and bacterial colonization in Hydra (Franzenberg et al.2012). The model annelid tubeworm, Hydroides elegans, undergoes metamorphosis in response to specific bacteria (e.g. Pseudomonas luteoviolacea) and is ideally suited to ask whether innate immunity is critical for bacterial stimulated metamorphosis (Ericson et al., 2019; Shikuma et al., 2014a). PATENT 5810.157405PCT / Shikuma-N6 RNAi has been used successfully in applications such as insect pest control and several human therapies targeting genetic diseases. However, difficulties in the delivery of RNAi molecules to cells, tissues or animals of interest limits the broad- sale usage of RNAi. While RNAi has been successfully employed for research, agricultural and therapeutic purposes, a current limitation of RNAi is the specific and prolonged delivery of double stranded RNA (dsRNA) to the organisms, tissues or cells of interest. Accordingly, there is a need for new technologies and vehicles for delivering RNAi molecules to cells, tissues and animals SUMMARY In alternative embodiments, provided are genetically engineered bacteria genetically engineered to have contained therein an expression vehicle or expression system comprising a non-coding RNA or a synthetic single-stranded non-coding DNA, or a plasmid or an expression vehicle or expression system capable of expressing the non-coding RNA or synthetic single-stranded DNA in a cell, or genetically engineered bacteria genetically engineered to have contained therein an expression vehicle or expression system capable of expressing a non- coding RNA such as RNAi or dsRNA, dsRNA, siRNA, shRNA, antisense oligonucleotides, ribozymes, aptamers, piRNAs, lncRNAs, or DNA fragments, wherein the genetically engineered bacteria comprises a targeting element or moiety or targeting mechanism capable of targeting a specific cell or tissue target, or the genetically engineered bacteria expresses extracellularly a targeting element or targeting moiety capable of targeting and then binding or adhering to a specific gene within a specific cell or tissue target, wherein the genetically engineered bacteria have either: (a) an endogenous or native nucleic acid transfer system to transfer the exogenous nucleic acid generated by the expression vehicle or expression system to the target cell; or (b) an exogenous or non-native transfer system inserted into the genetically engineered bacteria by genetic engineering. In alternative embodiments of genetically engineered bacteria as provided herein, or genetically engineered bacteria used in methods as provided herein: - the expression vehicle or expression system is or comprises an expression plasmid or engineered virus, or a Tn7 transposon system, CP25, T7, Ptac (Tac- PATENT 5810.157405PCT / Shikuma-N6 Promoter, abbreviated as Ptac), a tac vector, lac, trp, araBAD and / or λ PR (or Enterobacteria phage λ); the non-coding RNA comprises: a microRNA (miRNAs, or RNA molecule that binds complementary sequences of target mRNAs), wherein optionally the miRNA is or comprises a double-stranded RNA; a short hairpin RNAs (shRNAs, or an artificial RNA molecule with a tight hairpin turn that can be processed into siRNAs within cells) wherein optionally the shRNA is or comprises a double-stranded RNA; an antisense oligonucleotide (ASO, or a short, synthetic single-stranded DNA or RNA molecules designed to bind to specific mRNA sequences, blocking their translation or promoting degradation); an RNA aptamer (or an RNA molecule that can bind to specific proteins or other cellular targets, modulating their function); a ribozyme (or an RNA molecule with enzymatic activity capable of catalyzing specific biochemical reactions, such as RNA cleavage); a small activating RNA (saRNA, or a small double-stranded RNA that can upregulate gene expression by targeting promoter regions); a Piwi-interacting RNA (piRNA, or a small RNA that interacts with Piwi proteins and are primarily involved in silencing transposable elements and other genomic elements in the germline); and / or, a long Non-coding RNAs (lncRNA, or an RNA molecule longer than 200 nucleotides that does not code for proteins but can regulate gene expression at various levels); - the engineered virus is a genetically engineered virus; - the cell is a plant cell, or a crop cell, or an animal cell, or a human cell or a mucosal cell, or a gut mucosal cell; - the bacteria is non-pathogenic, innocuous or native to a target (or host) organism, or its environment, or is non-pathogenic, innocuous or native to a target’s microbiome; - the non-coding RNA or the synthetic single-stranded non-coding DNA can kill or neutralize or render non-pathogenic: a harmful insect or pathogen (optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen, PATENT 5810.157405PCT / Shikuma-N6 wherein optionally the vector borne pathogen is: Malaria, Dengue, Yellow Fever, West Nile Virus, Zika or Chikungunya), a bacterial or fungal infection, and optionally the viral pathogen is HIV-1, Poliovirus or Hepatitis C; - the non-coding RNA or the synthetic single-stranded non-coding DNA can kill or neutralize a pathogen in or on a: fin fish, mollusks (molluscs), or any organism in Crustacean aquaculture (optionally including: roundworms, nematodes, copepods, mites, and viruses that infect these organisms); - the non-coding RNA or the synthetic single-stranded non-coding DNA can treat or ameliorate, or slow the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease; - the non-coding RNA or the synthetic single-stranded non-coding DNA can treat or ameliorate, or slow the progress of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA targets an oncogene; and / or - the genetically engineered bacteria is isolated from or is derived from: an animal microbiome, optionally a gut microbiome, or a human microbiome, or an insect microbiome, or a plant microbiome; or, an environmental bacteria. In alternative embodiments, provided are cells comprising or having contained therein a genetically engineered bacteria as provided herein; and optionally the cell is a eukaryotic cell, optionally an animal, a plant cell, or a human cell. In alternative embodiments, provided are methods for administering or delivering a non-coding RNA or the synthetic single-stranded non-coding DNA to an organism or an individual in need thereof, comprising administering or delivering to the individual in need thereof a genetically engineered bacteria as provided herein, or a cell as provided herein. In alternative embodiments of methods as provided herein: - the genetically engineered bacteria or the cell is delivered or administered orally or implanted or delivered or administered rectally, or delivered or administered by inhalation, and optionally when the genetically engineered bacteria or the cell is delivered or administered orally or implanted or administered rectally or delivered or administered by inhalation the genetically engineered bacteria or the cell is formulated in an implant, a liquid, a powder, a tablet, a pill, a gel, a geltab, a PATENT 5810.157405PCT / Shikuma-N6 lyophilate or a freeze-dried material or formulation, an aerosol or a spray, and optionally the liquid is a sterile water or saline; - the genetically engineered bacteria or the cell is administered or delivered to: any animal, optionally any domesticated, farm, lab or wild animal, or to a human, a multicellular plant, any Mollusca, optionally any Porifera, Annelida, Crustacea, Echinoidea, and including oysters, clams, abalones, and mussels, optionally any Bivalvia, or any Pteriomorphia, or any animal of the group: Anomiidae (saddle oysters), Dimyidae (dimyarian oysters), Ostreidae (true oysters), Placunidae (windowpane oysters), Pteriidae (feather oysters), Spondylidae (spiny oysters), or any Anomioidea, Ostreida, Ostreoidea, Pectinoidea or Pterioidea and the like; or any Pteriomorphia (marine mussel), Palaeoheterodonta (freshwater mussel) or Heterodonta (zebra mussel); or any Atlantic Hard-Shell Clams (Mercenaria mercenaria), Soft-shell clams (Mya arenaria), Manila clams (Venerupis philippinarum), Surf clams (Spisula solida), Pacific razor clams (Siliqua patula), Atlantic Jackknife clam (Ensis leei) or Pacific Geoduck (Panopea generosa), or any abalone of the genus Haliotis. any Crustacea, optionally any crab, lobster, krill, shrimp, prawn, and crayfish, including for example any animal of the clades Oligostraca, Multicrustacea, and Allotriocarida, any animal of the group: Brachyura (True Crab), Lithodidae (King crab), Pseudothelphusidae (Freshwater crabs), Potamoidea (Freshwater crabs), Portunoidea (Swimming crabs), Trapexioidea (Coral crabs), Dromioidea (Sponge crab), Decapoda (Decopods), Nephropidae (Lobster), Palinuridae (Spiny Lobster), Scyllaridae (Slipper lobster), Dendrobranchiata (Prawns), Pleocyemata (Shrimp and Crayfish), and Euphausiacea (Krill), any invertebrate coral reef organism, optionally any organism of the phylum Cnidaria, optionally any jellyfish, anemones, stony corals, soft corals, fan corals, optionally any animal of the subclasses Aurelia (moon jelly), Nematostella (starlet sea anemone), Cassiopea (upside- PATENT 5810.157405PCT / Shikuma-N6 down jellyfish), Aiptasia (anemone), Heliporacea (crystalline octocoral), Alcyonaecea (soft coral), Scleractinia (stony corals), and Antipatharians (black coral), any Porifera, optionally any sponge classes including Demospngiae, Hexactinellida (Glass sponges), Calcarea (Calcareous sponges), or Homoscleromorpha, optionally any Xestospongia muta (Giant Barrel Sponge), Spheciospongia vesparium (Loggerhead sponge), Irchnia felix (Brown Branching sponge), Aplysinia fulva (Yellow Rope), Ircinia campana (Vase), Hippospongia lachne (Sheepswool sponge), Spongia barbara (Bath sponge), Spongia barbara dura, and Spongia graminea, any Annelida, optionally any Annelid class organism, optionally any Polychaeta, Clitellata, Machaeridia, and subphylum Sipuncula, and / or any kelp, or any large brown algae or seaweeds that make up the order Laminariales, including Agaraceae, Akkesiphycaceae, Alariaceae, Aureophycaceae, Chordaceae, Laminariaceae, Lessoniaceae, Pseudochordaceae, also including Chlorophyta (green algae), Phaeophyceae (brown algae), Phaeothamniophyceae, Chrysophyceae (gold algae), Rhodophyta (red algae); - the delivering or administering of the non-coding RNA or the synthetic single-stranded non-coding DNA to the organism or the individual in need thereof, results in or causes the killing, rendering non-pathogenic or neutralizing of a harmful insect or a pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen; and / or - the delivering or administering of the non-coding RNA or the synthetic single-stranded non-coding DNA to the individual in need thereof, results in or causes treating or ameliorating, or slowing the progress of, or decreasing or ameliorating the symptoms of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is a cancer or tumor, Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease. In alternative embodiments, provided are uses of genetically engineered bacteria as provided herein, or a cell as provided herein, for killing, rendering non- PATENT 5810.157405PCT / Shikuma-N6 pathogenic or neutralizing: a harmful insect or pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen. In alternative embodiments, provided are uses of genetically engineered bacteria as provided herein, or a cell as provided herein, for treating or ameliorating, or decreasing or ameliorating the symptoms of, or slowing the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease. In alternative embodiments, provided are uses of genetically engineered bacteria as provided herein, or a cell as provided herein, for treating or ameliorating, or slowing the progress of, or decreasing or ameliorating the symptoms of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA targets an oncogene. In alternative embodiments, provided are genetically engineered bacteria as provided herein, or a cell as provided herein, for use in killing, rendering non- pathogenic or neutralizing: a harmful insect or pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen. In alternative embodiments, provided are genetically engineered bacteria as provided herein, or a cell as provided herein, for use in treating or ameliorating, or slowing the progress of, or decreasing or ameliorating the symptoms of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease. In alternative embodiments, provided are genetically engineered bacteria as provided herein, or a cell as provided herein, for use in treating or ameliorating, or slowing the progress of, or decreasing or ameliorating the symptoms of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA targets an oncogene. The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications, American Type Culture Collection (ATCC) deposits and NCBI reference sequences cited herein are hereby expressly incorporated by reference in their entireties for all purposes. PATENT 5810.157405PCT / Shikuma-N6 DESCRIPTION OF DRAWINGS The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims. FIG.1A-K illustrates gene-specific knockdown of Hydroides via bacteria feeding and RNA interference: FIG.1A schematically illustrates an exemplary model of RNA interference bacterial feeding in Hydroides; the RNAi plasmid is carried by a feeder bacterium that Hydroides can utilize as a food source but does not stimulate metamorphosis, and the tubeworm’s RNA-Induced Silencing Complex (RISC) cleaves the dsRNA into small RNA fragments (siRNA), leading to targeted mRNA degradation; FIG.1B graphically illustrates expression (FPKM) of Hydroides RNAi genes from a transcriptome at 6-days post-fertilization; FIG.1C illustrates a hybridization chain reaction (HCR) fluorescent micrograph of RNA interference deficiency-1 (SID-1) and Argonaute-1 gene expression; FIG.1D graphically illustrates bacterial growth curve of P. mandapamensis bacteria WT and transfected with GFP dsRNA plasmid; FIG.1E graphically illustrates Hydroides metamorphosis results after 24 hour bacterial exposure to metamorphosis-associated contractile structures (MACs), filtered sea water (FSW), Phaeobacter mandapamensis, Vibrio fortis, and Vibrio harveyi; FIG.1F illustrates Differential Interference Contrast (DIC) images of a-tubulin RNAi knock down after 24 hour bacterial feeding of dsRNA alpha-tubulin (a-tubulin) and dsRNA; FIG.1B graphically illustrates the percent of larvae with a ciliary band fed dsRNA a-tubulin or dsRNA GFP; FIG.1H illustrates an image of anti a-tubulin antibody stain in Hydroides fed dsRNA a-tubulin or dsRNA gfp for 24 hours; FIG.1I graphically illustrates counts of ciliary band expression of anti-alpha- tubulin antibody stain in Hydroides fed dsRNA a-tubulin or dsRNA GFP for 24 hours; PATENT 5810.157405PCT / Shikuma-N6 FIG.1J illustrates an image of fluorescent in-situ of a-tubulin mRNA in Hydroides fed dsRNA a-tubulin or dsRNA gfp for 24 hours; and FIG.1E graphically illustrates quantitative-PCR of alpha-tubulin knock down against gfp. Values were normalized against the 18S housekeeping gene; as discussed in detail in Example 1, below. FIG.2A-D illustrates MyD88 knockdown inhibits metamorphosis: FIG.2A illustrates images of DIC micrographs of Hydroides larvae after 24 hour feeding of P. mandapamensis pDS-HeMyD88 (left) or P. mandapamensis pDS- gfp (right); FIG.2B graphically illustrates percent of larvae showing metamorphosis in Myd88 dsRNA and GFP dsRNA exposed larvae (n=100, 3 biological replicates); FIG.2C illustrates images of HCR of MyD88 gene in dsRNA MyD88 dsRNA fed larvae (left) and GFP dsRNA fed larvae (right) after 5 minute MAC induction. MyD88 expression is in turquoise, DNA (DAPI) is in grey; and FIG.2D graphically illustrates percent of larvae with MyD88 signal (n=30, 3 biological replicates) in MyD88 dsRNA fed larvae versus GFP dsRNA; as discussed in detail in Example 1, below. FIG.3A-L illustrate MyD88 activates immune and developmental genes upon the stimulation of metamorphosis by bacteria: FIG.3A illustrates images of RUNT expression in dsRNA GFP fed Hydroides larvae. FIG.3B illustrates images of RUNT expression in dsRNA MyD88 fed Hydroides larvae. FIG.3C graphically illustrates Larval counts of RUNT expression (n=30, 3 biological replicates). FIG.3E illustrates images of Immunoglobulin expression in dsRNA GFP fed Hydroides larvae. FIG.3E illustrates images of Immunoglobulin expression in dsRNA MyD88 fed Hydroides larvae. FIG.3F graphically illustrates Larval counts of Immunoglobulin expression (n=30, 3 biological replicates). FIG.3G illustrates images of NHR2 expression in dsRNA GFP fed Hydroides larvae. PATENT 5810.157405PCT / Shikuma-N6 FIG.3H illustrates images of NHR2 expression in dsRNA MyD88 fed Hydroides larvae. FIG.3I graphically illustrates Larval counts of NHR2 expression (n=30, 3 biological replicates). FIG.3J illustrates images of Cytokine expression in dsRNA GFP fed Hydroides larvae. FIG.3K illustrates images of Cytokine expression in dsRNA MyD88 fed Hydroides larvae; and, FIG.3L graphically illustrates Larval counts of Cytokine expression (n=30, 3 biological replicates), as discussed in detail in Example 1, below. FIG.4A-C illustrate Hydroides larvae feed on genetically modified marine bacteria: FIG.4A schematically illustrates an exemplary model of double stranded RNA (dsRNA) delivery via bacterial feeding to Hydroides larvae; FIG.4B graphically illustrates Hydroides metamorphosis after 24 hour bacterial exposure to MACs (Metamorphosis-Associated Contractile structures), artificial sea water (ASW), Photobacterium mandapamensis strains 4.11 and 4.16, Pseudoalteromonas piratica, Vibrio harveyi, and Vibrio fortis; FIG.4B illustrates images of micrographs of Hydroides larvae fed P. mandapamensis 4.11 wild-type (left image) or P. mandapamensis 4.11 mRuby-tagged bacteria (right image); dashed line outlines stomach (st) and anal vesicle (av) in Hydroides larvae; as discussed in detail in Example 1, below. FIG.5 illustrates Table 2, as described in detail in Example 1, below. FIG.6A graphically illustrates Hydroides larval responses when fed Photobacterium mandapamensis strains 4.11 and 4.16, E. coli HT115, Vibrio harveyi or Isochrysis galbana algae; eror bars represent standard deviation; FIG.6A graphically illustrates bacterial growth curve of P. mandapamensis 4.11 bacteria WT and P. mandapamensis 4.11 transformed with pDS-gfp dsRNA plasmid; PATENT 5810.157405PCT / Shikuma-N6 FIG.6C-E graphically illustrate representative immunofluorescence images of larvae fed P. mandapamensis 4.11 (FIG.6C) pDS-gfp, (FIG.6D) pDS-αtubulin or (FIG.6E) pDS-CA1 using an anti-αtubulin antibody. as discussed in detail in Example 1, below. FIG.7A-I illustrate gene-specific knockdown in Hydroides via bacteria feeding and RNA interference: FIG.7A illustrates images of DIC micrographs of Hydroides larvae after 24- hour feeding P. mandapamensis 4.11 with pDS-gfp, pDS-αtubulin, or pDS-CA1, the white arrow indicates the location of ciliary band loss; FIG.7B graphically illustrates percent larvae fed P. mandapamensis pDS-gfp or pDS-αtubulin with ciliary band after 24 hours; FIG.7C graphically illustrates counts of larvae with CA1 HCR expression 24 hours after feeding P. mandapamensis pDS-gfp or pDS-CA1; FIG.7D illustrates an image of HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-gfp after 24 hours, and lower left is 3.4x zoomed in panel of ciliary band and lower right is 3.4x zoomed in panel of CA1 gland for pDS-gfp exposed larvae; FIG.7E illustrates an image of HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-αtubulin after 24 hours, and lower left is 3.4x zoomed in panel of ciliary band and lower right is 3.4x zoomed in panel of CA1 gland for pDS-αtubulin exposed larvae; FIG.7F illustrates an image of HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-CA1 after 24 hours, and the lower left is 3.4x zoomed in panel of ciliary band and lower right is 3.4x zoomed in panel of CA1 gland for pDS-CA1 exposed larvae; FIG.7G graphically illustrates quantitative PCR (qPCR) of the Hydroides αtubulin gene in larvae fed P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 after 24 hours; FIG.7H graphically illustrates qPCR of the Hydroides CA1 gene in larvae fed P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 after 24 hours; and FIG.7I graphically illustrates counts of larvae with anti-αtubulin staining after feeding P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 for 24 hours, as discussed in detail in Example 1, below. PATENT 5810.157405PCT / Shikuma-N6 FIG.8 graphically illustrates Hydroides require MyD88 for bacterial pathogen defense: Hydroides larvae were exposed to artificial sea water (ASW), P. mandapamensis pDS-gfp or P. mandapamensis pDS-MyD88 for 24 hours; subsequently, larvae were exposed to Pseudomonas aeruginosa PA14 and scored for survival over 8 hours; the Y-axis represents % survival at each timepoint on the x-axis (0, 2, 4, 6, and 8 hours); error bars represent standard deviation, as discussed in detail in Example 1, below. Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION In alternative embodiments, provided are genetically engineered bacteria having contained therein an expression vehicle comprising a non-coding RNA or a synthetic single-stranded non-coding DNA, or a plasmid or an expression vehicle capable of expressing the non-coding RNA or synthetic single-stranded DNA in a cell, wherein the genetically engineered bacteria comprises a targeting element or moiety (for example, a nucleic acid targeting element or moiety) capable of targeting a specific gene within a specific cell or tissue target, or the genetically engineered bacteria expresses extracellularly a targeting element or targeting moiety capable of targeting and then binding or adhering to a specific gene within a specific cell or tissue target. In alternative embodiments, provided are methods of using genetically engineered bacteria as provided herein for administering or delivering a non-coding RNA or a synthetic single-stranded non-coding DNA to an organism or an individual in need thereof, wherein the administering or delivering is for treating or ameliorating infections, diseases, cancers and the like. This invention utilizes a promising strategy for genetically manipulating animals, including humans, called RNA interference (RNAi). The invention described here details methods, proof-of-concept data and examples of using environmental bacteria as a delivery vehicle for non-coding RNA (such as RNAi or dsRNA) molecules. Because some environmental bacteria are innocuous or naturally occurring, they could serve as an effective strategy for non-coding RNA molecule delivery. Such methods could allow a broader usage of non-coding RNA (such as RNAi or dsRNA) in previously inaccessible applications and markets. PATENT 5810.157405PCT / Shikuma-N6 Provided herein are novel methods and tools to genetically engineer environmental and / or microbiome bacteria to produce and deliver dsRNA to a plant or an animal for non-coding RNA (such as RNAi or dsRNA) treatment. Here, we provide exemplary methods and data for the genetic manipulation of a marine Photobacterium sp. bacteria to express dsRNA and feeding to a model marine invertebrate (Hydroides elegans tubeworms) for specific gene knockdown of cellular structure (tubulin) and immune system (MyD88) genes. These methods and data demonstrate the effectiveness of using non-coding RNA treatment by delivering dsRNA using genetically engineered bacteria, such as environmental and / or microbiome bacteria, and this genetic engineering allows the expression and delivery of non-coding RNA machinery for the genetic manipulation of target animals or plants of interest. The novel methods and tools to genetically engineer environmental and / or microbiome bacteria to produce and deliver non-coding RNA (such as RNAi or dsRNA) to a plant or an animal for non-coding RNA (such as RNAi or dsRNA) treatment have research, agricultural, aquaculture and biomedical applications. In alternative embodiments, environmental bacteria are used in aquacultures to provide a superior delivery vehicle of non-coding RNA (such as RNAi or dsRNA) machinery to organisms, where the non-coding RNA (such as RNAi or dsRNA) machinery targets viruses in aquaculture animals and plants. For example, in alternative embodiments, methods as provided herein are used to treat viral diseases such as herpes virus, such as herpes virus in oysters, which untreated has a $6B USD economic cost (Cain, 2022). Although non-coding RNA (such as RNAi or dsRNA) as been shown to successfully protect animals from viral diseases, the delivery of non- coding RNA machinery to the animal remains challenging. In alternative embodiments, methods as provided herein have biomedical applications, for example, using and delivering one of the non-coding RNA-based drugs that have received FDA approval (Traber and Yu, 2023). Use of methods as provided herein overcomes significant challenges in the delivery of non-coding RNA machinery for non-coding RNA-based therapeutics. In alternative embodiments, methods as provided herein using environmental or microbiome bacteria provides a superior delivery mechanism over viral vectors and PATENT 5810.157405PCT / Shikuma-N6 lipid-based nanoparticles for non-coding RNA-based therapies for plants, animals and humans. In alternative embodiments, methods as provided herein genetically engineer environmental or microbiome bacteria to advance the effectiveness of non-coding RNA therapy by delivering non-coding RNA (for example, dsRNA) through bacteria that may be innocuous or native to the host organism, its environment or microbiome through the use of a broad range bacterial plasmids. In alternative embodiments, methods as provided herein provide advantages to previously used methods in genetic engineering including TALENS and CRISPR for site-directed gene manipulation; TALENS (transcription activator-like (TAL) effector nucleases) and CRISPR (clustered regularly interspaced short palindromic repeats) technology target DNA and can create genetic changes in DNA in the host cell or organism; and in contrast, RNAi reduces RNA expression, without making changes to the DNA. In alternative embodiments, methods as provided herein delivery methods for RNAi are more straightforward than TALENS or CRISPR. While the machinery or genes encoding TALENS and CRISPR must be injected or delivered to the cell of interest, in alternative embodiments of methods as provided herein RNAi is administered by superficial soaking of plants or animals or through microbiome bacteria. Transferring or delivering non-coding RNA to a target or host cell or organism In alternative embodiments, methods as provided herein are practiced using bacteria that have endogenous, inherent, or wild-type, capabilities to transfer nucleic acid from the bacteria to a target cell. For example, in one alternative embodiments an Agrobacterium tumefaciens is used, where this bacterium which has a large tumor-inducing (Ti) plasmid: and upon detecting a wounded plant cell, the A. tumefaciens bacterium uses a specialized molecular syringe called a Type IV Secretion System (T4SS) to inject a segment of the Ti plasmid's DNA, called T-DNA, into the plant cell. In alternative embodiments, methods as provided herein genetically modify Agrobacterium tumefaciens to contain an expression vector which expresses a desired nucleic acid (such as a nucleic acid such as a dsRNA, siRNA, shRNA, antisense oligonucleotide, ribozyme, aptamer, piRNAs, lncRNA or DNA fragment and the like) to a host cell using its Type IV Secretion System (T4SS). PATENT 5810.157405PCT / Shikuma-N6 In alternative embodiments, a Rhizobium etli or Bartonella henselae bacterium is used: Bartonella henselae and Rhizobium etli also have a T4SS system that can inject nucleic acids into targeted host cells, and alternative embodiments this invention exploits the T4SS system to inject a desired nucleic acid into a host cell. In alternative embodiments, an Escherichia coli and other enterobacteria like Shigella use Type III Secretion Systems (T3SS) to inject effector proteins into host cells, and alternative embodiments this invention exploits the T3SS system to inject a desired nucleic acid into a host cell. In alternative embodiments, methods as provided herein expand the number and types of bacteria that could be used for non-coding RNA (for example, dsRNA or RNAi) delivery to a target cell or organism, and in alternative embodiments methods as provided herein are used to deliver the non-coding RNA (for example, dsRNA or RNAi) using bacteria that do not have an endogenous nucleic acid transfer mechanism, but rather have engineered therein a nucleic acid transfer mechanism, for example, the following list provides alternative mechanisms to engineer bacteria as provided herein to deliver non-coding RNA (for example, RNAi), and bacteria as provided herein either naturally (as a wild type) or are genetically engineered to comprise any of or at least one of the following nuclec acid transfer machinery: (1) E. coli have been engineered to deliver dsRNA to a number of organisms including: nematodes (C. elegans) for basic research (Timmons and Fire, 1998); fin fish to cause male to female conversion (Gao et al., 2024); and, shrimp for virus control (Thammasorn et al., 2013), accordingly, bacteria as provided herein are genetically engineered to comprise RNA (such as siRNA, RNAi or dsRNA) delivery systems as described by Timmons and Fire, 1998, Gao et al., 2024, or Thammasorn et al., 2013; (2) Microbiome bacteria that are native to the honeybee or insect host and deliver RNA interference machinery for parasite or disease prevention (Leonard et al., 2020). https: / / www.lens.org / lens / patent / 100-378-528-041- 856 / frontpage?l=en), accordingly, bacteria as provided herein are genetically engineered to comprise RNA (such as siRNA, RNAi or dsRNA) delivery systems as described by Leonard et al., 2020; (3) Bacteria native to the mosquito gut (Serratia fonticola) have been engineered to deliver RNAi machinery (Ding et al., 2023a), accordingly, PATENT 5810.157405PCT / Shikuma-N6 bacteria as provided herein are genetically engineered to comprise RNA (such as such as siRNA, RNAi or dsRNA) delivery systems as described by Ding et al., 2023a; (4) Probiotic bacteria (Lactobacillus plantarum) have been used to target viral diseases in shrimp aquaculture (Thammasorn et al., 2017) ), accordingly, bacteria as provided herein are genetically engineered to comprise targeting systems as described by Thammasorn et al., 2017; and (5) siRNA has been delivered via E. coli, Salmonella, Bacillus bacteria for therapeutic delivery, see USPN 10,987,432 B2, accordingly, bacteria as provided herein are genetically engineered to comprise RNA (such as siRNA, RNAi or dsRNA) delivery systems as described by USPN 10,987,432 B2. In alternative embodiments, the genetically engineered bacteria described herein may be further engineered to comprise structural elements that facilitate direct transfer of nucleic acids, including but not limited to dsRNA, siRNA, shRNA, antisense oligonucleotides, ribozymes, aptamers, piRNAs, lncRNAs, or DNA fragments, into a host cell or organism. In alternative embodiments these nucleic acid transfer structural elements are naturally encoded by the bacterium (endogenous to the bacteria). In alternative embodiments, the bacteria do not have an endogenous nucleic acid transfer mechanism, so these bacteria are genetically modified to contain an exogenous nucleic acid transfer mechanism from another species (for example, a Type I, Type II, and Type VII nucleic acid injection system), or contain an exogenous nucleic acid transfer mechanism that is partially or entirely a synthetic construct. In alternative embodiments, direct transfer of nucleic acids is effected through mechanisms of physical injection, vesicle encapsulation, secretion, or cell–cell contact. For example, an alternative embodiment comprises use of a secretion system that represents one broad category of delivery structures, such as a Type III secretion system; this Type III system operate as syringe-like nanomachines to translocate protein effectors directly into host cells. In some embodiments, these systems are modified to carry nucleic acid–binding proteins fused to dsRNA or siRNA, enabling translocation of therapeutic nucleic acids alongside effector proteins. In some PATENT 5810.157405PCT / Shikuma-N6 embodiments, a Type IV secretion system is used, and a Typie IV system is exemplified by the conjugation machinery of Agrobacterium tumefaciens, may be engineered to transfer plasmids, double-stranded RNA, or short hairpin RNA constructs into eukaryotic target cells. Type VI secretion systems, which are widespread in Gram-negative bacteria and operate as contractile injection devices, can be modified to puncture host cells and inject RNA molecules or RNA–protein complexes. In some embodiments, other secretion systems are used, including Type I, Type II, and Type VII, which optionally are modified or repurposed or engineered to export nucleic acids either freely into the extracellular milieu, for loading into vesicles, or for direct uptake at the cell surface. By expressly including secretion systems of all types (I through VII), methods as provided herein encompass both canonical and non-canonical bacterial secretion machinery capable of nucleic acid transfer. In some embodiments, bacteria that have endogenous Type I, Type II, and Type VII nucleic acid transfer systems are used to practice methods as provided herein. In addition to bacterial secretion systems, vesicle-based delivery mechanisms provide another structural embodiment used in methods as provided herein for direct transfer of nucleic acid from a bacteria to a target cell. In some embodiments bacteria that naturally produce outer membrane vesicles (OMVs) are used, or these bacteria also can be engineered to encapsulate therapeutic RNAs and deliver them into target host cells. Genetic modifications can result in the fusion of RNA-binding motifs, such as MS2 coat proteins or arginine-rich peptides, to proteins enriched in vesicles, thereby enhancing selective loading of nucleic acid cargo. In addition to OMVs, other vesicle-like systems such as bacterially derived lipid nanoparticles, minicells, or extracellular vesicles are employed to practice methods as provided herein. These vesicles, whether natural or synthetic, may be modified with ligands or targeting moieties to facilitate uptake by specific host cells. In alternative embodiments, bacteria that produce membrane nanotubes are used to practice methods as provided herein, where the membrane nanotubes act as physical conduits for material transfer between cells, and in some embodiments these nanotubes may be engineered to shuttle RNA or DNA molecules directly into PATENT 5810.157405PCT / Shikuma-N6 eukaryotic host cells. In alternative embodiments, bacteria are engineered de novo to produce membrane nanotubes to practice methods as provided herein. In alternative embodiments Contractile Injection Systems (CIS) are used to to practice methods as provided herein. For example, CIS are bacteriophage tail–like nanomachines that can puncture cell membranes and deliver protein effectors. . In alternative embodiments, bacteria are engineered de novo to produce Contractile Injection Systems (CIS) to practice methods as provided herein. In alternative embodiments, these nanomachines are modified to encapsulate a nucleic acid such as a dsRNA, siRNA, or DNA fragments for injection into host cells. In alternative embodiments genetic engineering targets and modifies sheath proteins, capsid-like chambers, or spike complexes, some or all of which can be adapted to bind and encapsulate therapeutic nucleic acids for direct cytoplasmic delivery. In some embodiments this mechanism of action is particularly advantageous because it bypasses endocytic uptake pathways and enables efficient injection directly into the cellular compartment of interest. In alternative embodiments surface display and fusion protein approaches provide further alternatives in practicing methods as provided herein. In such embodiments, bacteria are engineered to express RNA-binding proteins or peptides on their outer membrane, including arginine-rich motifs, polycationic domains, or CRISPR-associated RNA-binding proteins. By tethering therapeutic nucleic acids at the bacterial surface, these constructs allow for delivery upon intimate contact with a host cell, ensuring localized transfer of the nucleic acid payload. This strategy can be combined with secretion or vesicle release to further improve delivery efficiency. CRISPR-based structural elements may also be engineered as delivery platforms. For example, Cas9, Cas12, or Cas13 proteins complexed with guide RNAs can be packaged into bacterial secretion systems, vesicles, or contractile nanomachines, providing a programmable means of nucleic acid delivery. In such embodiments, the bacteria not only deliver nucleic acids but also provide precision gene regulation, silencing, or activation functions within the target host cell. This dual structural and functional role further strengthens the therapeutic utility of the invention. In alternative embodiments, symbiotic and probiotic bacteria themselves serve as carriers of delivery systems to practice methods as provided herein. In these embodiments, bacteria native to the host microbiome, or environmental bacteria that PATENT 5810.157405PCT / Shikuma-N6 naturally colonize the target organism, are genetically engineered to harbor one or more of the above structural features. In alternative embodiments these genetically engineered bacteria are used to colonize the host’s gut, mucosal surfaces, or other tissues and serve as persistent producers and delivery agents of therapeutic nucleic acids. In alternative embodiments, these genetically engineered bacterial symbionts utilize combinations of secretion systems, vesicle-mediated transfer, contractile injection, and surface display simultaneously, creating robust and redundant delivery pathways. For example, engineered bacteria may release nucleic acids in vesicles that are secreted through Type I secretion systems, or contractile injection systems may be used in tandem with vesicle-mediated transfer to maximize efficiency and specificity. Thus, in alternative embodiments bacteria are genetically engineered not only to produce therapeutic nucleic acids but also to comprise at least one structural delivery element, including secretion systems (Types I through VII), vesicles such as OMVs, lipid nanoparticles, minicells, or nanotubes, contractile injection systems, surface-displayed fusion proteins, CRISPR-associated complexes, and symbiont- based carriers. In alternative embodiments delivery elements may be natural, engineered, or synthetic, and may be used individually or in combination. In this way, the invention provides broad and flexible coverage over all known and foreseeable mechanisms by which bacteria can be engineered to transfer nucleic acids into host cells, thereby ensuring that alternative designs, whether using canonical systems, synthetic variants, or future-discovered nanostructures, remain within the scope of the invention. The embodiments described above are intended to supplement and further define the scope of the claims already directed to genetically engineered bacteria comprising an expression system for producing a non-coding RNA or synthetic single-stranded DNA. In particular, the structural elements disclosed herein — including secretion systems of all types (I through VII), vesicle-based delivery mechanisms (OMVs, lipid nanoparticles, minicells, extracellular vesicles, and membrane nanotubes), contractile injection systems, surface-displayed RNA-binding fusion proteins, CRISPR-associated delivery complexes, and symbiotic or probiotic carriers — are expressly contemplated as additional features that may be present in the genetically engineered bacteria as set forth in the claims. PATENT 5810.157405PCT / Shikuma-N6 In certain embodiments, the bacteria as claimed are further engineered to comprise one or more of these structural elements to effectuate the delivery or transfer of the therapeutic nucleic acid to a host cell or tissue. In some embodiments, the claims are intended to cover bacteria wherein the structural delivery element is native (or endogenous) to the bacterium, introduced from another organism, or wholly synthetic. In further embodiments, the claims encompass bacteria configured with two or more delivery mechanisms in combination, thereby broadening efficiency, specificity, and redundancy of nucleic acid transfer. Accordingly, the claims should be understood to extend not only to bacteria producing and carrying therapeutic nucleic acids, but also to bacteria further comprising any structural delivery apparatus, whether natural, engineered, or synthetic, that is capable of facilitating the transfer of such nucleic acids into a target cell, tissue, or organism. These exemplary embodiments ensure that alternative secretion or transfer modalities, hybrid designs, and future-discovered bacterial delivery systems are within the scope of genetically engineered bacteria and methods as provided herein.Aquaculture Applications In alternative embodiments, methods as provided herein are used in aquaculture industries, which are heavily affected by pathogens and viruses decreasing production. In some cases these pathogens can wipe out whole stocks of animals, such as white spot syndrome in shrimp. Additionally, viruses may be passed on to the consumer such as norovirus from filter feeding mollusks (molluscs). The economic cost of seafood illness is estimated to be $350 million annually in the United States (Ralston et al., 2011). Therefore, methods as provided herein provide the aquaculture industry with a needed pathogen control method that can specifically and effectively target these pathogens with minimal off target effects. In alternative embodiments, methods as provided herein use bacterial feeding RNAi technology to solve this problem by engineering microbiome or environmentally safe bacteria to deliver RNAi to the organism and provide protective immunity. In alternative embodiments, methods as provided herein are used in pathogen and parasite management for may different types of organisms and animals, including fin fish, mollusks (molluscs), and Crustacean aquaculture (including: roundworms, nematodes, copepods, mites, and viruses that infect these organisms). Table 1 PATENT 5810.157405PCT / Shikuma-N6 provides a list of diseases and pests that are targeted with bacterial delivery RNAi technology as provided herein: Table 1: In alternative embodiments, methods as provided herein are used for pathogen and disease control, and in alternative embodiments methods as provided herein use RNAi to create beneficial phenotypes with aquaculture species, including genetic modification of Crustaceans to promote growth, sex determination, disease prevention, parasite prevention or any phenotype. In alternative embodiments, methods as provided herein use a variety of applications for delivery of non-coding RNA (for example, dsRNA or RNAi) machinery via environmental or microbiome bacteria, and can be used beyond aquaculture and marine ecosystems, including for example a therapeutic for human diseases and pathogens, a microbiome editing tool for many organisms, and a pesticide alternative for the agriculture industry. In alternative embodiments, methods as provided herein use symbiotic microbiome bacteria to carry non-coding RNA (for example, dsRNA or RNAi) to mucosal surfaces of gastrointestinal, respiratory, and genitourinary tracts, and allow for colonization of that surface by symbiotic microbiome bacteria for effective delivery of non-coding RNA (for example, dsRNA or RNAi) to specific tissues PATENT 5810.157405PCT / Shikuma-N6 (Martirosyan et al., 2014). In alternative embodiments, methods as provided herein overcome a major roadblock for non-coding RNA (for example, dsRNA or RNAi) delivery as a therapeutic by having the ability to maintain an association with the mucosal layer within the digestive tract or as native residents of epithelial / skin tissue and not be quickly cleared out of the body (González et al., 2013). Unlike nanoparticles, the native microbiome bacteria used in methods as provided herein are known mucosal surface residents with established roles within mucosal and digestive tract microbiomes. In some embodiments, therapeutic nucleic acids delivered to host (or target) cells using methods as provided herein are designed to address (treat, prevent or ameliorate) an infectious disease, for example, an infectious disease caused by a viral pathogen, for example, an infectious disease caused by human immunodeficiency virus (HIV), influenza viruses, any hepatitis virus (for example, hepatitis B and C viruses), a coronavirus (optionally a SARS-CoV-2), herpesviruses, noroviruses, papillomaviruses, and arboviruses (optionally) Zika, dengue, chikungunya, yellow fever, and West Nile virus. In some embodiments, therapeutic nucleic acids delivered to host (or target) cells using methods as provided herein are designed to address (treat, prevent or ameliorate) bacterial or fungal pathogens and infections caused by bacteria and / or fungi, including but not limited to infections by Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium tuberculosis, Candida albicans, Cryptococcus neoformans. In alternative embodiments, methods as provided herein are used for viral pathogen control and control of vector borne pathogens such as: Malaria, Dengue, Yellow Fever, West Nile Virus, Zika, and Chikungunya, which are all on the rise due to the warming global temperatures. These major human illnesses will affect millions of people. Malaria alone infects 228 million people a year (Dahmana and Mediannikov, 2020). In alternative embodiments, methods as provided herein provide RNAi editing of the gut microbiome of mosquitoes (Ding et al., 2023b) to control viral and pathogen disease outbreaks. In alternative embodiments, methods as provided herein have the ability to utilize native gut or environmental microbiota to carry RNAi machinery allows for an expanded use of these pathogen control strategies for all of the major mosquito borne diseases. PATENT 5810.157405PCT / Shikuma-N6 In alternative embodiments, methods as provided herein are used as a disease therapeutic using non-coding RNA (for example, dsRNA or RNAi) delivery to target gene expression in a highly specific manner. In alternative embodiments, methods as provided herein use non-coding RNA (such as RNAi) as a therapeutic to treat many viral infections including HIV-1, Poliovirus and Hepatitis C (Agrawal et al., 2003). In addition to viral infections, in alternative embodiments, methods as provided herein are used for other disease targets such as cancer oncoproteins, which that are responsible for normal cells turning into cancer cells. In alternative embodiments, methods as provided herein are used to treat genetic disorders linked to specific genes, and methods as provided herein target these genes directly to treat the genetic disorder, including treating for example: Huntington’s Disease, Parkinson’s Disease, and Alzheimer's Disease (Seyhan, 2011). In alternative embodiments, methods as provided herein use delivery of RNAi via microbiome or environmental bacteria to treat human diseases and viral infections, thus making it a powerful and highly specific therapeutic.In alternative embodiments, methods as provided herein deliver therapeutic nucleic acids to a target cell, for example, methods as provided herein can deliver RNAi molecules (for example, siRNA, shRNA, dsRNA), antisense oligonucleotides, CRISPR guide RNAs and associated non-coding RNAs, ribozymes, aptamers, and DNA fragments; where in alternative embodiments once delivered to the target cell the delivered nucleic acid is designed (has the structure and / or sequence) to silence, activate, edit, or otherwise modulate host (target cell) gene expression. In alternative embodiments, nucleic acids delivered to target cells using bacteria and / or methods as provided herein are used in oncology applications, for example, to treat, ameliorate or prevent a tumor or a cancer, including for example, silencing of an oncogene, wherein the oncogene comprises: BCL2 (to treat for example, Chronic lymphocytic leukemia (CLL) or follicular lymphoma), mutant TP53, HER2 (ERBB2) (to treat breast cancer), Ras Family (KRAS, HRAS, NRAS) (to treat pancreatic cancer, lung cancer, or colorectal cancer), Myc (MYC) (to treat for example, Burkitt lymphoma), ABL1 (BCR-ABL1) (to treat . chronic myeloid leukemia (CML)), Src (SRC) (to treat colon cancer and breast cancer). In alternative embodiments, nucleic acids delivered to target cells using bacteria and / or methods as provided herein are used to treat, ameliorate or prevent a PATENT 5810.157405PCT / Shikuma-N6 genetic disorder, for example, Huntington’s disease, cystic fibrosis, Duchenne muscular dystrophy, fragile X syndrome, hemophilia. In alternative embodiments, nucleic acids delivered to target cells using bacteria and / or methods as provided herein are used to treat, ameliorate or prevent a neurological or neural degenerative condition or disease such as for example: a neuropathy, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), Multiple Sclerosis (MS), Spinocerebellar Ataxias (SCAs), Lewy Body Dementia (LBD). In alternative embodiments, nucleic acids delivered to target cells using bacteria and / or methods as provided herein are used to treat, ameliorate or prevent an autoimmune or inflammatory disease or condition, for example, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, multiple sclerosis, optionally by suppressing pro-inflammatory cytokines such as: Interleukin-1 beta (IL-1β), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6), IL-17, Interferon-gamma (IFN-γ), Interleukin-12 (IL-12), Granulocyte-Macrophage Colony- Stimulating Factor (GM-CSF). In alternative embodiments, nucleic acids delivered to target cells using bacteria and / or methods as provided herein are used to treat, ameliorate or prevent a metabolic or a cardiovascular disease such as: Peripheral Artery Disease (PAD), Coronary Artery Disease (CAD) or atherosclerosis, an ischemic stroke or hemorrhagic stroke, congestive heart failure, optionally by modulation of lipid metabolism, insulin sensitivity, or cardiac remodeling. Bacterial formulations and in vivo therapeutic delivery In alternative embodiments, as discussed above, genetically engineered bacteria are administered in vivo to an individual (a patient) in need thereof to treat, ameliorate or prevent a broad spectrum of conditions where modulation of host gene and / or protein expression confers a therapeutic effect of a clinical benefit. In alternative embodiments, the administered genetically engineered bacteria as provided herein are attenuated, have no or reduced virulence, are non-virulent and / or are non-pathogenic, for example, attenuated, non-virulent and / or non- pathogenic bacteria can be used as live-attenuated vaccine, where the bacteria deliver nucleic acids encoding immunogens. PATENT 5810.157405PCT / Shikuma-N6 In alternative embodiments, the administered genetically engineered bacteria as provided herein are lyophilized or freeze dried before in vivo administration. In alternative embodiments, the lyophilized or freeze dried administered genetically engineered bacteria as provided herein are administered in lyophilized or freeze dried state as a powder or inhalant, or, are reconstituted in a liquid (for example, saline or water) formulation. In alternative embodiments, the genetically engineered bacteria as provided herein are administered in vivo to an individual (a patient) in need thereof by oral, rectal, vaginal, nasal, intradermal, intrathecal, or pulmonary administration, or parenterally, including subcutaneous, intramuscular, intravenous and intradermal administration, or by inhalation. In alternative embodiments, the genetically engineered bacteria as provided herein are formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and / or carriers used to practice embodiments as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo applications. In practicing embodiments as provided herein, the genetically engineered bacteria as provided herein or as used in methods as provided herein can comprise a solution of genetically engineered bacteria disposed or formulated in a pharmaceutically acceptable carrier, for example, acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, formulations used to practice embodiments as provided herein are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques. The genetically engineered bacteria as provided herein used to practice methods as provided herein can comprise auxiliary substances as required to PATENT 5810.157405PCT / Shikuma-N6 approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of genetically engineered bacteria as provided herein in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results. The genetically engineered bacteria as provided herein used to practice methods as provided herein can be formulated in and delivered by the use of liposomes. In alternative embodiments, by using liposomes, particularly where the liposome surface carries ligands specific for target cells or organs, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo, in vitro or ex vivo application. Nanoparticles, Nanolipoparticles and Liposomes Provided are nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising genetically engineered bacteria used to practice the methods and embodiments as provided herein. Provided are multilayered liposomes comprising compounds used to practice embodiments as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No.20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein. In alternative embodiments, any delivery vehicle can be used to practice the methods as provided herein, e.g., to deliver genetically engineered bacteria as provided herein, or a genetically engineered bacteria used to practice methods as provided herein, to mammalian cells, e.g., in vivo, in vitro or ex vivo. For example, delivery vehicles comprising polycations, cationic polymers and / or cationic peptides, such as polyethyleneimine derivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No.20060083737. The pharmaceutical compositions and formulations as provided herein or as used in methods as provided herein comprising genetically engineered bacteria as provided herein can be administered for prophylactic and / or therapeutic treatments. PATENT 5810.157405PCT / Shikuma-N6 In therapeutic applications, genetically engineered bacteria are administered to a subject, for example, a human in need thereof, in an amount of the genetically engineered bacteria sufficient to cure, alleviate or partially arrest the clinical manifestations and / or its complications (a “therapeutically effective amount”). The amount of pharmaceutical formulation adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. In alternative embodiments, genetically engineered bacteria as provided herein are administered at a dosage of between about 103and 1012genetically engineered bacteria per ml. or per gram if a non-liquid formulation. The state of the art allows the clinician to determine the dosage regimen for each individual patient and disease or condition treated. Accordingly, methods as provided herein, and engineered bacteria provided herein and used in methods as provided herein, can produce (generate) therapeutic nucleic acids (for example therapeutic nucleic acids, for example, methods as provided herein can deliver RNAi molecules (for example, siRNA, shRNA, dsRNA), antisense oligonucleotides, CRISPR guide RNAs and associated non-coding RNAs, ribozymes, aptamers, and DNA fragments) in bacteria, which then actively target host cells through specific bacterial proteins, and thereby optionally can be used to treat, ameliorate or prevent a broad spectrum of conditions where modulation of host gene and / or protein expression confers a therapeutic effect of a clinical benefit. In alternative embodiments, methods and engineered bacteria provided herein and used in methods as provided herein are used as a pesticide, thus providing an alternative to current approaches to treating agriculture crops (for example, an alternative to the use of pesticides, which are expensive and have off target effects). In alternative embodiments, methods as provided herein provide for nucleic acid delivery (for example non-coding RNA (for example, RNAi molecules (for example, siRNA, shRNA, dsRNA), antisense oligonucleotides, CRISPR guide RNAs and associated non-coding RNAs, ribozymes, aptamers, and DNA fragments) nucleic acid) deliveryto specific target pathogen cells (optionally cells of harmful insects, PATENT 5810.157405PCT / Shikuma-N6 viral pathogens, and fungal infections) (see for example, Giudice et al., 2021), thus optionally positively influencing the growth and vitality of plants and crops without any effects to plants and crops themselves. In alternative embodiments, methods as provided herein deliver RNAi directly to the plants and crops to eliminate crop pests, and make the plants and crops disease resistant, thermotolerant, and increase crop yield. In alternative embodiments, methods as provided herein and engineered bacteria provided herein and used in methods as provided herein are used as an alternative non-coding RNAs regulating gene expression system; wherein, in addition to RNA interference, methods as provided herein deliver other non-coding RNAs to animals or plants, human or plant cells, and / or tissues or organisms, via microbiome or environmental bacteria. Exemplary non-coding RNAs delivered by methods to plant cells (or any cell, including any eucharyotic cell, such as a mammalian cell, such as a human cell) as provided herein include: ● microRNAs (miRNAs) – RNA molecules bind complementary sequences of target mRNAs. ● Short hairpin RNAs (shRNAs) – artificial RNA molecules with a tight hairpin turn that can be processed into siRNAs within cells. ● Antisense Oligonucleotides (ASOs) – short, synthetic single-stranded DNA or RNA molecules designed to bind to specific mRNA sequences, blocking their translation or promoting degradation. ● RNA Aptamers – RNA molecules that can bind to specific proteins or other cellular targets, modulating their function. ● Ribozymes – RNA molecules with enzymatic activity capable of catalyzing specific biochemical reactions, such as RNA cleavage. ● Small Activating RNAs (saRNAs) – small double-stranded RNAs that can upregulate gene expression by targeting promoter regions. ● Piwi-interacting RNAs (piRNAs) –small RNAs that interact with Piwi proteins and are primarily involved in silencing transposable elements and other genomic elements in the germline. ● Long Non-coding RNAs (lncRNAs) – a diverse class of RNA molecules longer than 200 nucleotides that do not code for proteins but can regulate gene expression at various levels. PATENT 5810.157405PCT / Shikuma-N6 Exemplary Mechanisms of dsRNA uptake and gene silencing Noting that the invention is not limited by any particular mechanism of action, in alternative embodiments: double stranded RNA (dsRNA) enters the animal through feeding of genetically engineered bacteria as provided herein that are producing dsRNA from a replicative plasmid. In alternative embodiments, the sequence of the dsRNA encoded is targets a gene of interest. Once within the animal's digestive tract the genetically engineered bacteria lyse releasing dsRNA into the intestine. The dsRNA is taken up by the intestinal cells by passive transport across the cell membrane, which can be mediated by the gene SID-1 which acts as a dsRNA-gated channel (Zhuang and Hunter, 2012). In alternative embodiments, when dsRNA enters the intestinal cells they are processed into small interfering RNAs (siRNAs) by an dsRNA specific RNAse-III-type endonuclease Dicer that cleaves the dsRNA into 20- 25 nucleotide siRNAs (Meister and Tuschl, 2004). The siRNAs then act as a guide for the RNA-induced silencing complex (RISC) to find complementary messenger RNA (mRNA) molecules. Once RISC binds to the corresponding mRNA sequence that matches the siRNA, the mRNA is degraded by the protein Argonaute2 which is contained in the RISC complex (Hammond, 2005; Hammond et al., 2001). Argonaute2 acts as a slicer, which binds and degrades the mRNA resulting in the silencing of that corresponding gene. Exemplary bacteria In alternative embodiments, provided are genetically engineered bacteria genetically engineered to have contained therein an expression vehicle or system for non-coding RNA delivery to an organism or individual in need thereof, the expression vehicle or system comprising a non-coding RNA or a synthetic single-stranded non- coding DNA, or a plasmid or an expression vehicle capable of expressing the non- coding RNA or synthetic single-stranded DNA in a cell, and methods of making and using them. In alternative embodiments, bacteria used to generate genetically engineered bacteria as provided herein, and to practice methods as provided herein, can be from (or derived from) any human or environmental bacteria (optionally a microbiome bacterium or a human or animal probiotic bacterium), or a bacterium of the genus: Nereida, Roseobacter, Pseudoalteromonas, Vibrio, or Bacteroidetes (Alker et al., 2023b, 2023a; Rojas et al., 2020); PATENT 5810.157405PCT / Shikuma-N6 or a bacterium from a human probiotic bacterial genus, family, order or classes: Lactobacillales, Bifidobacteriales, Sphingomonadales, or Hyphomicrobiales (Gao et al., 2023); or an environmental bacteria from marine and soil microbiomes, optionally of the genus, family, order or classes of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Campylobacterales, Flavobacteriia, Bacilli, Acidimicrobiia, Actinomycetia, Cytophagia, and Chitinophagia (Aislabie and Deslippe, 2013; Martínez Cruz et al., 2012; Schulze et al., 2006), or a Roseobacter species have been identified as candidates for probiotic bacteria in aquaculture (Sonnenschein et al., 2021), or an E. coli strain used as a probiotic bacteria in humans (Ou et al., 2016). or for delivery of non-coding RNA to ocean-dwelling organisms, in alternative embodiment the bacteria can include Vibrio, Pseudomonas, Pseudoalteromonas, Lactobacillus, Bacillus, Photobacterium, and Enterococcus or Roseobacter species, many of these bacteria have been genetically modified previously (Alker et al., 2023a), and these bacteria can be used to delivery non-coding RNA to marine animals in aquaculture, for example, including shrimp, prawns, oysters, mussels, clams, and sea urchins, or can be used for for marine restoration, for example, including oysters, mussels, clams, sea urchins, corals, and sea cucumbers, or for delivery of non-coding RNA for human therapeutic purposes, the bacteria can include Lactobacillus, Bifidobacterium, Bacillus, Enterococcus, Pediococcus, Streptococcus, Akkermansia, Faecalibacterium, Clostridium, Propionibacterium, Roseburia, Eubacterium, Ruminococcus, Lactococcus, or Leuconostoc; in alternative embodiments, these bacteria and non-coding RNA are used to treat a number of human conditions including cancer, cardiovascular diseases, neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, genetic disorders like cystic fibrosis, Duchenne muscular dystrophy, and fragile X syndrome, viral infections including HIV, hepatitis B and C, and SARS-CoV-2, autoimmune diseases such as rheumatoid arthritis, lupus, and multiple sclerosis, metabolic disorders like diabetes, obesity, and rare metabolic conditions such as phenylketonuria, inflammatory diseases including Crohn’s disease and ulcerative colitis, and chronic conditions such as asthma and chronic obstructive pulmonary disease (COPD), PATENT 5810.157405PCT / Shikuma-N6 or for delivery of non-coding RNA for agriculture purposes, in alternative embodiments bacteria used include Bacillus, Pseudomonas, Azospirillum, Rhizobium, Enterococcus, Lactobacillus, Streptomyces, Paenibacillus, Frankia, Burkholderia, Azotobacter, Actinobacteria, Clostridium, Arthrobacter, Bradyrhizobium, Mesorhizobium, Serratia, Nostoc, Anabaena, Klebsiella, Micrococcus, Pantoea, Photorhabdus, Xanthomonas, Thiobacillus, Variovorax, or Acinetobacter; in alternative embodiments these bacteria and non-coding RNA are used for treatment of viral disease, bacterial disease, fungal disease, pest management or crop enhancement. Exemplary expression systems used with RNAi In alternative embodiments, genetically engineered bacteria as provided herein, and as used in methods as provided herein, are genetically engineered to have contained therein an expression vehicle or expression system capable of expressing a non-coding RNA or synthetic single-stranded DNA in a cell, for example, a bacterial cell. In alternative embodiments, expression systems used to produce non-coding RNA, include any plasmid or any one of: or a Tn7 transposon system, CP25, T7, Ptac (Tac-Promoter, abbreviated as Ptac), a tac vector, lac, trp, araBAD and / or λ PR (or Enterobacteria phage λ), or a plasmid or expression system as described in U.S. patent no.11,382,989. We have demonstrated that a Tn7 transposon system can be used for integration of genetic material into the bacterial genome (Alker et al., 2023b), and the Tn7 system can be used for an expression system to produce the non-coding RNA from the bacterial genome. While the invention is not limited by any particular mechanism of action, in alternative embodiments a plasmid inside of the bacterial cell is designed to produce double stranded RNA of whatever gene of interest placed into the vector, and when ingested by the host, the bacterial cell bursts inside the host and releases this double stranded RNA which then integrates into the tissues and cells of the organisms ( but is not targeted to a specific location or cell type). Whatever gene that double stranded RNA is encoding is then silenced (knocked down) in all cells / tissues that gene is already being expressed due to the organism's own defense system against double stranded RNA utilizing genes like SID-1, Argonaut and the like, because the organism now thinks a gene is on that shouldn't be; this is done routinely in E coli with C. elegans (terrestrial nematodes) for gene knockouts. PATENT 5810.157405PCT / Shikuma-N6 In alternative embodiments a unique aspect of processes as provided herein is the creation of plasmid machinery designed for marine bacteria that are receptive to the plasmid and are easily taken up by a diverse range of marine animals, allowing for bacterial fed gene silencing in marine animals which hasn't been done before. Examples of bacterial proteins that target human cells In alternative embodiments, provided are methods having bacteria transfer nucleic acids produced in the bacteria (such as a genetically engineered bacteria) to a target cell, which can be a mammalian cell, for example a human cell. In alternative embodiments, transfer of a nucleic acid produced in the bacteria can occur in vitro, in situ or in vivo. In alternative embodiments, the genetically engineered bacteria as described herein comprise bacterial surface proteins or adhesins capable of binding to and targeting cells such as plant, insect, human or animal cells, thereby enhancing or effecting delivery (for example, intracellular delivery) of therapeutic nucleic acids to or into the targeted cell. In alternative embodiments, the bacterial surface proteins or adhesins that are capable of binding to and targeting human or animal cells to effect delivery of therapeutic nucleic acids are endogenous to the delivering bacteria. In alternative embodiments, the bacterial surface proteins or adhesins that are capable of binding to and targeting a cell (for example, human, insect or animal cells) to effect delivery of therapeutic nucleic acids are exogenous to the delivering bacteria, and the delivering bacteria are genetically engineered to functionally express the nucleic acid- delivering bacterial surface proteins or adhesins. . In alternative embodiments, the bacterial surface proteins or adhesins that are capable of binding to and targeting a cell (for example, human, insect or animal cells) to effect delivery of therapeutic nucleic acids can be any mechanism known in the art for example, exemplary nucleic acid systems comprise: - invasins from Yersinia spp., which bind β1-integrins on mammalian epithelial cells and promote internalization; - internalin A (InlA) from Listeria monocytogenes, which engages E-cadherin (CDH1) on human epithelial cells; - internalin B (InlB) from L. monocytogenes, which binds and activates the Met (hepatocyte growth factor) receptor to trigger uptake; PATENT 5810.157405PCT / Shikuma-N6 - pertactin (PRN) from Bordetella pertussis, an autotransporter adhesin bearing an RGD motif that supports integrin-mediated adhesion to respiratory epithelia; - fibronectin-binding proteins (FnBPA / B) from Staphylococcus aureus, which bind fibronectin and thereby bridge to α5β1 integrin on host cells; - protein A (SpA) from S. aureus, which binds the Fc region of IgG and can be employed in engineered or attenuated forms for targeting; - Opa proteins from Neisseria gonorrhoeae, which specifically engage CEACAM receptors on human mucosal cells; - filamentous hemagglutinin (FHA) from B. pertussis, a major adhesin that mediates robust attachment to host respiratory epithelium; - Type IV Secretion System (T4SS) (encoded by the VirB / VirD4 genes) that acts like a molecular syringe, allowing bacteria to transport DNA and / or proteins across their membranes and into a target cell, that can be from Agrobacterium, or A. tumefaciens, or Bartonella henselae; and / or - Type VI Secretion System (T6SS) used by many bacteria to inject toxins into neighboring cells, both prokaryotic (other bacteria) and eukaryotic (host) cells, a functional analog of a bacteriophage tail comprising a long, hollow tube made of protein that is surrounded by a contractile sheath; - mammalian microvesicles: - mammalian extracellular vesicles and tunneling nanotubes (TNTs) (made of actin and allow for the direct transfer of cellular material from one cell to another without leaving the extracellular space), noting that TNTs are used by mammalian macrophages, dendritic cells, T-cells, B-cells, and natural killer (NK) cells, and cancer cells such as glioblastoma, lung, breast, ovarian, and pancreatic cancer cells; and / or - mammalian extracellular vesicles (EVs) such as exosomes. In alternative embodiments, the bacteria are engineered to express fragments, domains, synthetic derivatives, or functional homologs of these proteins configured to confer host-targeting capabilities without pathogenicity, and in yet further embodiments such proteins are fused to RNA-binding domains, secretion system components, or vesicle-associated proteins to couple host recognition with nucleic- acid delivery. PATENT 5810.157405PCT / Shikuma-N6 Products of manufacture and Kits Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein. Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and / or Detailed Description sections. As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such PATENT 5810.157405PCT / Shikuma-N6 documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court. Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims. The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples. EXAMPLES Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in PATENT 5810.157405PCT / Shikuma-N6 McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany. Example 1: Animal metamorphosis requires bacteria and the innate immune system This example demonstrates that methods as provided herein can be effectively used to deliver any non-coding RNA such as RNAi to animal and plant cells to modify phenotypes and treat various diseases and conditions. Interactions between microbes and animals are critical for host development, yet the mechanisms of how bacteria promote development are not fully understood. We investigated a host-microbe interaction involving a marine tubeworm, Hydroides elegans, that undergoes metamorphosis in response to stimulation by marine bacteria. By creating a novel marine bacteria mediated RNA interference approach, we analyzed the Hydroides innate immune system in response to Pseudoalteromonas luteoviolacea, a metamorphosis-inducing marine bacterium. Our findings show MyD88, a critical immune adaptor for the TLR4 pathway, is necessary for the stimulation of metamorphosis in response to bacteria. In addition to a developmental role, we show that MyD88 is necessary during pathogenic exposure to the pathogen Pseudomonas aeruginosa, showing that Hydroides utilizes immunity during both development and infection. We conclude that the innate immune system is essential during a beneficial host-microbe developmental interaction, providing the first detailed characterization of an innate immune pathway involved in a basal metazoan's metamorphic development process. Results RNA interference (RNAi) for gene knockdown in Hydroides. Bacteria expressing double stranded RNA (dsRNA) have been shown to be an effective delivery strategy for employing RNAi in bacterivorous nematodes (Grishok, 2005). Similarly, Hydroides larvae and adults filter food from the water column and their growth has been shown to be supported by feeding on bacteria (Gosselin & Qian, 2000). We therefore sought to develop a strategy for performing RNAi with Hydroides by feeding them environmental bacteria producing double stranded RNA (Figure 1A). By searching the genome and transcriptome (Shikuma et al.2016), we determined that Hydroides possesses and expresses the molecular machinery critical for RNAi (Figure 1B, Table S1). PATENT 5810.157405PCT / Shikuma-N6 Table S1. Genes required for RNA interference in Hydroides. Visualization of the anatomical location of the key RNAi genes Argonaute and Sid1 using Hybridization Chain Reaction (HCR) showed that both genes are expressed throughout the larval animal, especially in the gut (Figure 1C). We next tested a series of 6 bacteria for their ability to sustain the growth of Hydroides from zygote to mature larva (competence). Hydroides did not grow or survive when exposed to E. coli HT115, the strain used for RNAi through feeding in Caenorhabditis elegans nematodes (Kamath et al., 2001). However, Photobacterium mandapamensis strain 4.11 supported the growth of Hydroides to competence at a rate comparable to feeding the traditional algal food Isochrysis galbana (Table S2) (Nedved & Hadfield, 2008; Nesbit & Shikuma, 2023). Table S2. Bacteria supporting the growth and development of Hydroides PATENT 5810.157405PCT / Shikuma-N6 Survival and development rating; (-) no survival, no growth. (+) low survival, some growth. (++) moderate survival, moderate growth. (+++) complete survival, good growth. We found that P. mandapamensis 4.11 was amenable to genetic engineering (Alker et al., 2023), and growth of the bacterium containing a broad-host range plasmid was similar to the growth of P. mandapamensis 4.11 wild-type (Figure 1D). While some bacteria stimulate the metamorphosis of Hydroides larvae, P. mandapamensis 4.11 only supported growth and did not stimulate metamorphosis (Figure 1E). To determine whether Hydroides is amenable to RNAi gene knockdown via bacterial feeding, we transformed P. mandapamensis 4.11 with a broad-host-range plasmid (Alker et al., 2023; Leonard et al., 2020) containing a 180 bp fragment of the Hydroides alpha tubulin (α-tubulin) (TCONS_00033662) flanked by two constitutive CP25 promoters (pDS-Hetubulin). Upon feeding Hydroides with P. mandapamensis pDS-Hetubulin, we observed a loss of the swimming ciliary band, while Hydroides larvae fed P. mandapamensis expressing dsRNA targeting a 200bp fragment of GFP (pDS-gfp) that does not occur in Hydroides showed no loss of the ciliary band (Figure 1F and G). Immunofluorescence of larvae fed P. mandapamensis pDS-Hetubulin confirmed a loss of tubulin protein when compared to larvae fed P. mandapamensis pDS-gfp (Figure 1H and I). HCR and quantitative PCR confirmed a decrease in α- tubulin mRNA in Hydroides fed bacteria with dsRNA targeting α-tubulin (Figure 1J and K). These results indicate that Hydroides is amenable to RNAi via feeding of P. mandapamensis expressing dsRNA. Figure 1. Gene-specific knockdown of Hydroides via bacteria feeding and RNA interference. (A) Model of RNA interference bacterial feeding in Hydroides. The RNAi plasmid is carried by a feeder bacterium that Hydroides can utilize as a food source but does not stimulate metamorphosis. The tubeworm’s RNA-Induced Silencing Complex PATENT 5810.157405PCT / Shikuma-N6 (RISC) cleaves the dsRNA into small RNA fragments (siRNA), leading to targeted mRNA degradation. (B) Expression (FPKM) of Hydroides RNAi genes from a transcriptome at 6-days post-fertilization (Shikuma et al., 2016). (C) Hybridization chain reaction (HCR) fluorescent micrograph of RNA interference deficiency-1 (SID-1) and Argonaute-1 gene expression. (D) Bacterial growth curve of P. mandapamensis bacteria WT and transfected with GFP dsRNA plasmid. (E) Hydroides metamorphosis results after 24 hour bacterial exposure to metamorphosis-associated contractile structures (MACs), filtered sea water (FSW), Phaeobacter mandapamensis, Vibrio fortis, and Vibrio harveyi. (F) DIC images of a-tubulin RNAi knock down after 24 hour bacterial feeding of dsRNA alpha-tubulin (a-tubulin) and dsRNA (G) Percent of larvae with a ciliary band fed dsRNA a-tubulin or dsRNA GFP. (H) Anti a-tubulin antibody stain in Hydroides fed dsRNA a-tubulin or dsRNA gfp for 24 hours. (I) Counts of ciliary band expression of anti-alpha-tubulin antibody stain in Hydroides fed dsRNA a-tubulin or dsRNA GFP for 24 hours. (J) Fluorescent in-situ of a-tubulin mRNA in Hydroides fed dsRNA a-tubulin or dsRNA gfp for 24 hours. (K) Quantitative-PCR of alpha-tubulin knock down against gfp. Values were normalized against the 18S housekeeping gene. Results are an average of 3 biological replicates. Hydroides requires MyD88 to undergo metamorphosis in response to bacteria. We analyzed the genome of Hydroides and identified a comprehensive set of genes responsible for an innate immune response. The homology analysis conducted against human genes revealed significant similarities, indicating the conservation of innate immune pathways across species, including Toll-like receptor 4 (TLR4), Myeloid differentiation primary response 88 (MyD88), Interleukin-1 receptor-associated kinase (IRAK), TNF receptor-associated factor 3 (TRAF3), TGF-beta-activated kinase 1 (TAK1), Immunoglobulin, and IL17. All of these genes were found to be expressed in larval animals (Table S3), suggesting that Hydroides possess a complete innate immune system. Table S3. MyD88 / TLR Pathway Gene Homologs in Hydroides. PATENT 5810.157405PCT / Shikuma-N6 To test whether Hydroides uses its innate immune system to undergo metamorphosis in response to bacteria, we created pDS-MyD88 containing a 200 bp fragment of Hydroides MyD88 (TCONS_00085996, Table S3). We then feed Hydroides with P. mandapamensis pDS-MyD88 for 24 hours followed by stimulating the larvae to undergo metamorphosis with a known bacterial stimulant (MACs) from Pseudoalteromonas luteoviolacea (Ericson et al., 2019; Shikuma et al., 2014b). Upon stimulation by MACs, MyD88 knockdown animals were unable to undergo metamorphosis in response to MACs in comparison to control pDS-gfp knockdown animals (Figure 2A and B). Moreover, using HCR and quantitative PCR, we observed a decrease in MyD88 expression, while animals fed control bacteria targeting gfp showed a robust expression of MyD88 in the swimming ciliated band (Figure 2C and D). These results demonstrate that MyD88 is critical for Hydroides to undergo metamorphosis in response to bacteria. Figure 2. MyD88 knockdown inhibits metamorphosis: (A) DIC micrographs of Hydroides larvae after 24 hour feeding of P. mandapamensis pDS-HeMyD88 (left) or P. mandapamensis pDS-gfp (right) (B) Percent of larvae showing metamorphosis in Myd88 dsRNA and GFP dsRNA exposed larvae (n=100, 3 biological replicates). (C) HCR of MyD88 gene in dsRNA MyD88 dsRNA fed larvae (left) and GFP dsRNA fed larvae (right) after 5 minute MAC induction. MyD88 expression is in turquoise, DNA (DAPI) is in grey. (D) Percent of larvae with MyD88 signal (n=30, 3 biological replicates) in MyD88 dsRNA fed larvae versus GFP dsRNA. MyD88 is required for immune and developmental pathways during metamorphosis. To determine the effect of MyD88 knock down on both developmental and immune genes downstream of MyD88, we identified a set of two immune and two developmental genes that have robust expression in Hydroides larvae (Table S3 and 4). We identified a Hydroides immunoglobulin, which are ligand binding heterodimeric proteins that have important roles in immune recognition receptor function and antigen recognition in vertebrates (Buckley & Rast, 2015; PATENT 5810.157405PCT / Shikuma-N6 Dishaw et al., 2016; Schroeder & Cavacini, 2010). We identified a Hydroides inflammatory cytokine, which have been shown to play central roles in immune response mediation to pathogens through regulating inflammation and activation of specific immune cell types (Buckley et al., 2017; Cerami, 1992; Choghakhori et al., 2017; Hawiger, 2001; Hughes & Nibbs, 2018). RUNT is a developmental gene regulating multiple developmental pathways. In Drosophila, RUNT functions as a pair rule transcription factor for both embryonic pattern formation development and neuronal development (Canon & Banerjee, 2000). In mammals, the mammalian RUNT domain plays a central role in development of blood and bone (Wheeler et al., 2000). Finally, He-NHR2 is a nuclear hormone receptor gene previously described to be activated during Hydroides metamorphosis (Malter et al., 2022). Nuclear hormone receptors play important roles in growth, differentiation, reproduction and morphogenesis of vertebrates (Aranda & Pascual, 2001). After feeding for twenty- four hours on P. mandapamensis pDS-MyD88 or pDS-gfp, the gene expression of RUNT (Figure 3A-C), immunoglobulins (Figure 3D-F), and NHR2 (Figure 3G-I) were significantly downregulated in MyD88 dsRNA fed larvae after exposure to MACs. In contrast, cytokine production was not significantly affected by the MyD88 knock down (Figure 3J-L). These results indicate that MyD88 plays a role in both developmental and immune function during metamorphosis. Table S4. Hydroides immunity and developmental genes examined. Figure 3. MyD88 activates immune and developmental genes upon the stimulation of metamorphosis by bacteria: (A) RUNT expression in dsRNA GFP fed Hydroides larvae. (B) RUNT expression in dsRNA MyD88 fed Hydroides larvae. (C) Larval counts of RUNT expression (n=30, 3 biological replicates). (D) Immunoglobulin PATENT 5810.157405PCT / Shikuma-N6 expression in dsRNA GFP fed Hydroides larvae. E. Immunoglobulin expression in dsRNA MyD88 fed Hydroides larvae. (F) Larval counts of Immunoglobulin expression (n=30, 3 biological replicates). (G) NHR2 expression in dsRNA GFP fed Hydroides larvae. (H) NHR2 expression in dsRNA MyD88 fed Hydroides larvae. (I) Larval counts of NHR2 expression (n=30, 3 biological replicates). (J) Cytokine expression in dsRNA GFP fed Hydroides larvae. (K) Cytokine expression in dsRNA MyD88 fed Hydroides larvae. (L) Larval counts of Cytokine expression (n=30, 3 biological replicates). Bacterial feeding and RNA interference (RNAi) for gene knockdown in Hydroides. Bacteria expressing double-stranded RNA (dsRNA) have been shown to be an effective delivery strategy for employing RNAi in model invertebrates such as bacterivorous nematodes and freshwater planarians (23–25). Similarly, Hydroides larvae and adults filter food from the water column and their growth has been shown to be supported by feeding on bacteria (26). We therefore sought to develop a strategy for performing RNAi with Hydroides by feeding them environmental bacteria producing dsRNA (FIG.4A). By searching the genome and transcriptome (14), we determined that Hydroides possesses and expresses the molecular machinery critical for RNAi (Table 2, see FIG.5) (23). FIG.4 illustrates Hydroides larvae feed on genetically modified marine bacteria: FIG.4 (A) Model of double stranded RNA (dsRNA) delivery via bacterial feeding to Hydroides larvae. The RNA interference (RNAi) plasmid is carried by a feeder bacterium that serves as a food source but does not stimulate metamorphosis. The tubeworm’s RNA-Induced Silencing Complex (RISC) cleaves the dsRNA into small RNA fragments (siRNA), leading to targeted mRNA degradation. FIG.4 (B) Graph of Hydroides metamorphosis after 24 hour bacterial exposure to MACs (Metamorphosis-Associated Contractile structures), artificial sea water (ASW), Photobacterium mandapamensis strains 4.11 and 4.16, Pseudoalteromonas piratica, Vibrio harveyi, and Vibrio fortis. Data are generated from 3 independent experiments (n=3). Error bars represent standard deviation. FIG.4 (C) Micrograph of Hydroides larvae fed P. mandapamensis 4.11 wild- type or P. mandapamensis 4.11 mRuby-tagged bacteria. Dashed line outlines stomach (st) and anal vesicle (av) in Hydroides larvae. PATENT 5810.157405PCT / Shikuma-N6 To identify a strain of bacteria that serves as a food source without stimulating metamorphosis, we exposed Hydroides larvae to a series of five strains of marine bacteria. Of those bacteria tested, Photobacterium mandapamensis 4.11, P. mandapamensis 4.16, and Vibrio harveyi did not induce metamorphosis (27) (FIG. 4B). When tested for their ability to sustain the growth of Hydroides from zygote to mature (competent) larva, P. mandapamensis 4.11 supported larval growth to competency at a level comparable to the traditional algal food Isochrysis galbana (21, 28) (FIG.6A). Hydroides did not grow or survive when exposed to E. coli HT115, the strain used for RNAi through feeding in Caenorhabditis elegans nematodes (29). We found that P. mandapamensis 4.11 was amenable to genetic engineering, and the growth of P. mandapamensis 4.11 containing a broad-host-range plasmid (pDS-gfp) used for genetic manipulation was similar to the growth of P. mandapamensis 4.11 wild type (FIG.6AB). When fluorescently tagged with an mRuby protein, P. mandapamensis 4.11 could be visualized within the Hydroides larval gut (FIG.6C). To determine whether Hydroides is amenable to RNAi gene knockdown via bacterial feeding, we transformed P. mandapamensis 4.11 with a plasmid (30–32) containing a 180 bp fragment of the Hydroides αtubulin gene (TCONS_00033662, Table 2, FIG.5) flanked by two constitutive CP25 promoters (pDS-αtubulin). Upon feeding Hydroides with P. mandapamensis pDS-αtubulin, we observed a loss of the swimming ciliary band while Hydroides larvae fed P. mandapamensis expressing dsRNA targeting a 200 bp fragment of gfp (pDS-gfp), a gene that does not occur in the Hydroides genome, showed no loss of the ciliary band (FIG.7A-B). Visualization and quantification of αtubulin expression using Hybridization Chain Reaction (HCR) and quantitative PCR (qPCR) confirmed the knockdown of αtubulin along the Hydroides ciliary band (prototroch) (FIG.7D, FIG.7E, FIG.7G). To test the specificity of RNAi knockdown, we fed Hydroides larvae P. mandapamensis with an RNAi plasmid targeting a second gene, a 270 bp fragment of the Hydroides carbonic anhydrase 1 (CA1) gene (TCONS_00094394, pDS-CA1, Table 2, FIG.5), which is involved in tube building and expressed in a distinct anatomical location within the larval collar tissue, below the ciliary band (33). HCR and qPCR confirmed the knockdown of CA1 in larvae fed pDS-CA1 while the expression in larvae fed pDS- αtubulin or pDS-gfp was unaffected (FIG.7C, FIG.7F, FIG.7H). Corresponding αtubulin immunostaining was reduced in larvae fed P. mandapamensis pDS-αtubulin PATENT 5810.157405PCT / Shikuma-N6 compared to larvae fed P. mandapamensis pDS-gfp or pDS-CA1 (FIG.7I and FIG. 6C-E). These results indicate that Hydroides is amenable to gene-specific RNA interference via feeding of P. mandapamensis expressing dsRNA. FIG.6(A): Graph of Hydroides larval responses when fed Photobacterium mandapamensis strains 4.11 and 4.16, E. coli HT115, Vibrio harveyi or Isochrysis galbana algae. Larvae were scored over six technical replicates and three biological replicates (n=3). Error bars represent standard deviation. FIG.6(B): Bacterial growth curve of P. mandapamensis 4.11 bacteria WT and P. mandapamensis 4.11 transformed with pDS-gfp dsRNA plasmid. FIG.6 (C-E) Representative immunofluorescence images of larvae fed P. mandapamensis 4.11 (C) pDS-gfp, FIG.6 (D) pDS-αtubulin or (E) pDS-CA1 using an anti-αtubulin antibody. FIG.7A-G: Gene-specific knockdown in Hydroides via bacteria feeding and RNA interference. FIG.7(A) DIC micrographs of Hydroides larvae after 24-hour feeding P. mandapamensis 4.11 with pDS-gfp, pDS-αtubulin, or pDS-CA1. White arrow indicates the location of ciliary band loss. FIG.7 (B) Percent larvae fed P. mandapamensis pDS-gfp or pDS-αtubulin with ciliary band after 24 hours. Data are generated from 3 independent experiments (n=3) with an average of 300 larvae scored for each treatment (****P<0.0001, calculated using a chi-square test with Yates’ correction). Error bars represent standard deviation. FIG.7 (C) Counts of larvae with CA1 HCR expression 24 hours after feeding P. mandapamensis pDS-gfp or pDS-CA1. Data are generated from an average of 30 larvae scored for each of 3 independent experiments (n=3) (****P<0.0001, calculated using a chi-square test with Yates’ correction). Error bars represent standard deviation. FIG.7 (D) HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-gfp after 24 hours. Lower left 3.4x zoomed in panel of ciliary band and lower right 3.4x zoomed in panel of CA1 gland for pDS-gfp exposed larvae. FIG.7 (E) HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-αtubulin after 24 hours. Lower left 3.4x PATENT 5810.157405PCT / Shikuma-N6 zoomed in panel of ciliary band and lower right 3.4x zoomed in panel of CA1 gland for pDS-αtubulin exposed larvae. FIG.7 (F) HCR of αtubulin (turquoise), CA1 (magenta) and DNA (DAPI, gray) in larvae fed P. mandapamensis pDS-CA1 after 24 hours. Lower left 3.4x zoomed in panel of ciliary band and lower right 3.4x zoomed in panel of CA1 gland for pDS-CA1 exposed larvae. FIG.7 (G) Quantitative PCR (qPCR) of the Hydroides αtubulin gene in larvae fed P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 after 24 hours. Data are generated from approximately 500 larvae in each of 3 technical replicates for each of 3 independent experiments (n=3). Fold change values calculated using DDCT method (Letters represent One-way ANOVA and Tukey post hoc test results, P<0.001). Error bars represent standard deviation. FIG.7 (H) qPCR of the Hydroides CA1 gene in larvae fed P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 after 24 hours. Data are generated from approximately 500 larvae in each of 3 technical replicates for each of 3 independent experiments (n=3). Log2 fold change values calculated using DDCT method (Letters represent One-way ANOVA and Tukey post hoc test results, P<0.001). Error bars represent standard deviation. FIG.7 (I) Counts of larvae with anti-αtubulin staining after feeding P. mandapamensis pDS-gfp, pDS-αtubulin or pDS-CA1 for 24 hours. Graph is an average of 3 biological replicates (n=3) with 30 larvae per replicate per treatment (chi-square test with Yates’ correction, ****P<0.0001 comparing pDS-gfp to pDS- αtubulin, n.s. comparing pDS-gfp to pDS-CA1). Error bars represent standard deviation. MyD88 plays dual roles in pathogen response and metamorphosis. To determine if MyD88 plays a canonical role in immune pathway activation, we exposed larvae fed P. mandapamensis with pDS-gfp or pDS-MyD88 to the bacterial pathogen, Pseudomonas aeruginosa PA14, which has been reported to cause disease in plants, nematodes, insects, mice and humans (35–37). When exposed to four of six densities (OD600 of 0.19, 0.38, 0.75, 1.5) of P. aeruginosa PA14, the survival of Hydroides fed P. mandapamensis pDS-MyD88 was significantly reduced (FIG.8). In contrast, the survival of Hydroides fed P. mandapamensis pDS-gfp showed no difference with larvae exposed to artificial seawater alone. These results demonstrate that MyD88 is critical for survival of Hydroides in response to a bacterial pathogen. PATENT 5810.157405PCT / Shikuma-N6 FIG.8 illustrates Hydroides require MyD88 for bacterial pathogen defense: Hydroides larvae were exposed to artificial sea water (ASW), P. mandapamensis pDS-gfp or P. mandapamensis pDS-MyD88 for 24 hours. Subsequently, larvae were exposed to Pseudomonas aeruginosa PA14 and scored for survival over 8 hours. The Y-axis represents % survival at each timepoint on the x-axis (0, 2, 4, 6, and 8 hours). Graph is an average of 3 biological replicates (n=3) with 30 larvae per replicate per treatment. One-way ANOVA was performed against all six treatments at each individual optical density averaged across the 8 hours. A Tukey post hoc test was performed to test for differences between each treatment. Letters denote statistically different treatments. Error bars represent standard deviation. Conclusions Based on our results, we propose that the MyD88 / TLR innate immune pathway is a critical feature of the life-history transition of metamorphosis in response to bacteria. The sensing of and response to stimulatory bacteria via the immune system is likely critical for the life history and ecology of animals throughout the animal kingdom. Materials and Methods Hydroides Collection and Maintenance Hydroides elegans animals were collected from the Quivera Basin, San Diego CA and kept as described by Ericson, eLife (2019). Gametes were spawned from adult animals and fertilized in 1L beakers with 0.45 µM filtered artificial seawater (ASW). Larvae were reduced to 5 larvae per mL at 24 hours post fertilization and fed iso- crysis algae at an average of 60,000 cells per mL. Water changes were performed daily. Larvae were considered competent between days 6 and 8 as described in (Nesbit & Shikuma, 2023). Metamorphosis Assays Metamorphosis assays were performed in a 96 well plate with competent larvae spawned from three different mate pairs, each mate pair was treated as a separate biological replicate. Treatments included a 1:100 dilution of MACs and ASW as a control. Each treatment included three technical replicates. Larvae were exposed for 24 hours and juveniles in each treatment were counted. An average of the three technical replicates in each treatment was taken and then averaged across the three bio replicates. Standard deviation was also calculated for each treatment. PATENT 5810.157405PCT / Shikuma-N6 RNAi Plasmid Construction and Cloning Golden Gate Assembly was performed to create the RNAi plasmids. The backbone consisted of a GFP dropout flanked by BsaI cut sites (Table S5). Table 3. Plasmids used in this work. The type 2 part consisted of a forward CP25 constitutive promoter and a reverse rnpB-T1 terminator (Leonard et al.2020). The type 4 part consisted of a reverse CP25 promoter and forward rpoC terminator. The coding sequences were created as type 3 parts, with a 300-400 bp fragment of the targeted gene. GOLDEN GATE PATENT 5810.157405PCT / Shikuma-N6 ASSEMBLY™ was performed with NEBRIDGE GOLDEN GATE ASSEMBLY KIT™ for BsaI-HF v2 with 50 fmol of each plasmid used in the reaction. Reaction was run with the following thermocycler program 37 ºC for 1 minute, 16 ºC for 1 minute, repeat cycles 1-230x, 60 ºC for 5 minutes. The assemblies were electroporated into SM10 λpir cells, confirmed by colony PCR (ECONOTAQ PLUS GREEN™, LGC Biosearch) with internal primers (Table S6) and then electroporated into MFDλpir cells for conjugation. Table S4. Plasmids used in this work. Plasmid Plasmid Type Description Marker Origin Source No. RNAi plasmid producing double As pDS-gfp stranded RNA GenR RSF1010 described matching gfp (Green herein Lantern). RNAi plasmid As pNJS10 pDS-MyD88 Type producing double 14 1-8 stranded RNA GenR RSF1010 described matching MyD88. herein RNAi plasmid As pNJS10 Type producing double pDS-αtubulin GenR RSF1010 described 25 1-8 stranded RNA herein matching αtubulin. RNAi plasmid As Type producing double pDS-CA1 GenR RSF1010 described 1-8 stranded RNA herein matching CA1. CP25 promoter on pNJS10 forward strand with pBTK150 Type 2 CamR ColE1 (2) 06 rnpB terminator on the reverse strand rpoC terminator pNJS10 forward strand with pBTK151 Type 4 CamR ColE1 (2) 07 CP25 promoter on the reverse strand pNJS63 pBTK402 Type 8 rfp dropout KanR RSF1010 (3) 6 As pNJS92 pMMK818 Type 8 Tn7 backbone KanR R6K described 1 herein pBTK402_GF Type Backbone vector As pNJS10 P_ 2,3,4 where type 2,3,4 parts RSF1010 described 13 Dropout_RSF Dropo drop out during herein 1010 GenR ut assembly. PATENT 5810.157405PCT / Shikuma-N6 KanR pNJS94 Mini Tn7 pTac pMMK819 promoter with G (Vector) As 7 1-8 FP GenR R6K described and T7 terminator (insertion) herein Mini Tn7 pTac KanR pNJS94 Type promoter with (Vector) As 9 pMMK820 1-8 mRuby and T7 GenR R6K described terminator (insertion) herein Table S5. Strains used in this work. Strain no. Strain Genotype Source ΔdapA::(erm-pir) NJS604 Escherichia coli MFD λpir ΔrecAthi thr leu (4) tonA lacY supE pNJS033 Escherichia coli SM10 λpir Tc::Mu Km λpir (5) F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lacUV5 (6) promoter -T7 polymerase MT004 Photobacterium mandapamensis 4.11 StR (7) MT038 Photobacterium mandapamensis 4.11 tn7_ptac_mRuby As described herein Maloy NJS653 Vibrio fortis WT - As described herein NJS005 Pseudoalteromonas luteoviolacea HI1 StR (9) NJS863 Pseudomonas aeruginosa PA14 - Lisa Racki PATENT 5810.157405PCT / Shikuma-N6 Table 5. Primers used. PATENT 5810.157405PCT / Shikuma-N6 Production of MAC extracts Metamorphosis-associated contractile structure (MAC) purification was carried out as described in (Rocchi et al., 2019). Pseudoalteromonas luteoviolacea HI1 was struck out to single colonies onto Sea Water Tryptone (SWT) media (35.9 g / L Instant Ocean, 2.5 g / L tryptone, 1.5 g / L yeast extract, 1.5 mL / L glycerol, and 15 g / L agar grown at 25 C overnight. A single isolated colony was inoculated into a 5 mL SWT culture and grown shaking overnight at 200 rpm in 25 C. The culture was then inoculated 1:100 into a 50mL SWT culture shaking overnight at 200 rpm in 25 C. The 50mL culture was centrifuged at 4C for 20 minutes at an rpf of 4,800. The supernatant was then removed and the pellet was resuspended in 5mL of extraction buffer (20 mM Tris Base, 1 M NaCl, pH 7.5) and then centrifuged again at 4C for 20 minutes at an rpf of 4,800. This was repeated once more for a total of three times. After the final centrifugation, 3mL was removed from the top of the supernatant. The MAC extract was then tested on competent larvae at a dilution of 1:100 to determine MAC efficacy. MACs were used within 24 hours of production. Bacterial Bi-parental Mating E. coli donor strains MFDλpir containing RNAi replicative plasmids were grown under antibiotic selection in LB (Miller, BD Difco) with 20 µg / mL of gentamicin and 0.3 mM of Diaminopimelic acid (DAP). A single colony of MFDλpir containing RNAi plasmids was inoculated and grown overnight in liquid culture at 37 ºC shaking PATENT 5810.157405PCT / Shikuma-N6 at 200 rpm. Marine bacteria strains Photobacterium mandapamensis 4.11 and Photobacterium mandapamensis 4.16 were inoculated with 3 colonies in Marine Broth (MB) 2216 (BD Difco) and grown overnight in liquid culture at 25 ºC shaking at 200 rpm. Cultures were spun down (4000 x g for 10 minutes). All supernatant was removed from each culture and the pellets were resuspended in 150 µL of MB. Donor and recipient cultures were mixed (200 µL total volume) and spotted in 50 µL spots onto MB agar plates and incubated overnight at 25 ºC. The next day, each individual spot was scraped up and resuspended in 250 µL of MB media. To wash the spots this process was repeated with the final resuspension in 100 µL. The full 100 µL was then plated onto MB agar plates with 200 µg / mL of gentamicin. Colonies were PCR screened for the plasmid using internal primers. Larval RNAi exposures Photobacterium mandapamensis 4.11 containing RNAi plasmids (Table S4) were struck out on a plate with Gentamicin and grown at 25˚C overnight. A single colony was then inoculated into 25mL of MB media with Gentamicin. After 16 hours of growth, cultures were spun down into a pellet and washed three times with 5mL of ASW, optical density was then quantified using a NANODROP™. Larvae were fed bacteria at an OD of 0.25 for 24 hours in FSW. Larvae were checked for abnormalities and competency before metamorphosis induction. Larvae were starved 24 hours prior to feeding to ensure adequate feeding on bacteria. Larval RNA extractions Competent larvae were exposed to a 1:100 dilution of MACS or ASW. Roughly 500 larvae were collected at 30 minutes and 2 hours of exposure and preserved in RNA Later (0.5M EDTA, 1M Sodium Citrate) for RNA extraction. Samples were kept at - 80 C and then placed on ice for RNA extraction. RNA was extracted from larvae using the MACHEREY NAGEL RNA EXTRACTION KIT™ (Catalog # 740955.50). Samples were kept on ice and washed with 1mL of RNase free PBS and spun down with a manual centrifuge at 100 rpm for 30 seconds. This was repeated twice before larvae were lysed with the RNA extraction lysis buffer. After lysis and desalting and DNase removal step was performed, RNA was then washed and eluted in RNase free water. RNA was quantified using a NANODROP™, quality RNA was used with 260 / 230 and 260 / 280 ratios between 1.8 and 2.1. cDNA Synthesis and Quantitative PCR PATENT 5810.157405PCT / Shikuma-N6 cDNA synthesis was performed on RNA samples using NEB biolabs PROTOSCRIPT II FIRST STRAND CDNA SYNTHESIS KIT™ (Catalog # E6560S). Each cDNA reaction was standardized to synthesize 1ug of RNA. Reactions were carried out at 42 C for 1 hour followed by five minutes at 85oC. cDNA was kept at -80oC for 48 hours. Quantitative PCR was performed with exon-exon primers designed in PRIMER 3™. Genes analyzed were FOS, IL17, FREP and TLR4. Alpha Tubulin was chosen as a housekeeping control and 18S was used as an internal control. Each 20 uL qPCR reaction was created using 10µL of PRIMA qMAX™ mix (Catalog # PR2120-H-S) , 50 ng of cDNA and 3.5 µL of forward and reverse primers. Ct values were normalized to the Ct values of 18S. Ct values from experimental treatments were subtracted from control treatments to calculate a delta Ct value. A delta-delta CT value was then calculated using the formula 2–∆∆Ct.. Graphs of these results were created using PRISM V10™. Hybridization Chain Reaction Whole mount embryo HCRs were carried out as described in (CITE urchin molecular instruments protocol) in 1.5mL tubes. Probes were designed by Molecular Instruments using mRNA coding sequences from Hydroides transcriptome. Amplifiers and probe washing buffers were purchased from Molecular Instruments. Larvae were exposed to a 1:100 dilution of MAC extracts for five and thirty minutes. Larvae were then treated in 6.5% MgCl to relax larvae and then washed twice with FSW. Larvae were fixed in 4% PFA (2.5mL 16% PFA, 1 mL 10x PBS Buffer, 6.5 mL RNAse free H20) overnight at 4C on a nutator. Samples were washed in 5X SSCT (10mL of 20x sodium citrate sodium citrate (SSC), 400 uL 10% Tween 20, 44.5 mL RNASE free H20) and probes were added to hybridization buffer at a concentration of 1pM per gene, Samples were pre-hybed for 30 minutes at 37C before probes were added. The following day, samples were washed four times in intervals of 30 minutes with probe wash buffer. Hairpins were heat shocked at 95oC for 90 seconds and snap cooled for 30 minutes at room temperature. Hairpins and amplifier buffer were added to samples and incubated overnight at 37oC. The following day, samples were washed 4 times with 5X SSCT in thirty-minute intervals and mounted in PROLONG ANTIFADE DIAMOND MOUNTANT™ with DAPI (# P36961). Samples were imaged the same day on a ZEISS AXIO OBSERVER.Z1™ inverted microscope PATENT 5810.157405PCT / Shikuma-N6 equipped with an AXIOCAM 506™ mono camera and NEOFLUAR10X / 0.3 PH1 20X™ objective. References Agrawal N, et al.2003. 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Aquaculture 324–325:303–306. doi:10.1016 / j.aquaculture.2011.10.018 Sarathi M, et al.2008. Oral Administration of Bacterially Expressed VP28dsRNA to Protect Penaeus monodon from White Spot Syndrome Virus. Mar Biotechnol 10:242–249. doi:10.1007 / s10126-007-9057-6 Seyhan AA.2011. RNAi: a potential new class of therapeutic for human genetic disease. Hum Genet 130:583–605. doi:10.1007 / s00439-011-0995-8 Sun Y, et al.2018. Integration of RNAi and RNA-seq Reveals the Immune Responses of Epinephelus coioides to sigX Gene of Pseudomonas plecoglossicida. Front Immunol 9. doi:10.3389 / fimmu.2018.01624 Thammasorn T, et al.2017. Probiotic bacteria (Lactobacillus plantarum) expressing specific double-stranded RNA and its potential for controlling shrimp viral and bacterial diseases. Aquacult Int 25:1679–1692. doi:10.1007 / s10499-017-0144-z Thammasorn T, et al.2013. Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon. Antiviral Res 100:202–206. doi:10.1016 / j.antiviral.2013.08.005 Timmons L, Fire A.1998. Specific interference by ingested dsRNA. Nature 395:854–854. doi:10.1038 / 27579 Tirasophon W, et al.2005. Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA. Biochemical and Biophysical Research Communications 334:102–107. doi:10.1016 / j.bbrc.2005.06.063 Traber GM, Yu A-M.2023. RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies. J Pharmacol Exp Ther 384:133–154. doi:10.1124 / jpet.122.001234. PATENT 5810.157405PCT / Shikuma-N6 A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

PATENT 5810.157405PCT / Shikuma-N6 WHAT IS CLAIMED IS:

1. A genetically engineered bacteria genetically engineered to have contained therein an expression vehicle or expression system comprising a non-coding RNA or a synthetic single-stranded non-coding DNA, or a plasmid or an expression vehicle or expression system capable of expressing the non-coding RNA or synthetic single-stranded DNA in a cell, or the genetically engineered bacteria are genetically engineered to have contained therein an expression vehicle or expression system capable of expressing a nucleic acid exogenous to the bacteria, and the nucleic acid comprises a non-coding RNA such as RNAi or dsRNA, dsRNA, siRNA, shRNA, antisense oligonucleotides, ribozymes, aptamers, piRNAs, lncRNAs, or DNA fragments, wherein the genetically engineered bacteria comprises a targeting element or moiety or targeting mechanism capable of targeting a specific cell or tissue target, or the genetically engineered bacteria expresses extracellularly a targeting element or targeting moiety capable of targeting and then binding or adhering to a specific gene within a specific cell or tissue target, wherein the genetically engineered bacteria have either: (a) an endogenous or native nucleic acid transfer system to transfer the exogenous nucleic acid generated by the expression vehicle or expression system to the target cell; or (b) an exogenous or non-native transfer system inserted into the genetically engineered bacteria by genetic engineering.

2. The genetically engineered bacteria of claim 1, wherein the expression vehicle or expression system is or comprises an expression plasmid or engineered virus, or a Tn7 transposon system, CP25, T7, Ptac (Tac-Promoter, abbreviated as Ptac), a tac vector, lac, trp, araBAD and / or λ PR (or Enterobacteria phage λ).

3. The genetically engineered bacteria of claim 1, wherein the non-coding RNA comprises: a microRNA (miRNAs, or RNA molecule that binds complementaryPATENT 5810.157405PCT / Shikuma-N6 sequences of target mRNAs), wherein optionally the miRNA is or comprises a double-stranded RNA; a short hairpin RNAs (shRNAs, or an artificial RNA molecule with a tight hairpin turn that can be processed into siRNAs within cells) wherein optionally the shRNA is or comprises a double-stranded RNA; an antisense oligonucleotide (ASO, or a short, synthetic single-stranded DNA or RNA molecules designed to bind to specific mRNA sequences, blocking their translation or promoting degradation); an RNA aptamer (or an RNA molecule that can bind to specific proteins or other cellular targets, modulating their function); a ribozyme (or an RNA molecule with enzymatic activity capable of catalyzing specific biochemical reactions, such as RNA cleavage); a small activating RNA (saRNA, or a small double-stranded RNA that can upregulate gene expression by targeting promoter regions); a Piwi-interacting RNA (piRNA, or a small RNA that interacts with Piwi proteins and are primarily involved in silencing transposable elements and other genomic elements in the germline); and / or, a long Non-coding RNAs (lncRNA, or an RNA molecule longer than 200 nucleotides that does not code for proteins but can regulate gene expression at various levels).

4. The genetically engineered bacteria of claim 2, wherein the engineered virus is a genetically engineered virus.

5. The genetically engineered bacteria of any of claims 1 to 4, wherein the cell is a plant cell, or a crop cell, or an animal cell, or a human cell or a mucosal cell, or a gut mucosal cell.

6. The genetically engineered bacteria of any of claims 1 to 6, wherein the bacteria is non-pathogenic, innocuous or native to a target (or host) organism, or its environment, or is non-pathogenic, innocuous or native to a target’s microbiome.PATENT 5810.157405PCT / Shikuma-N6 7. The genetically engineered bacteria of any of claims 1 to 6, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA can kill, neutralize or render non-pathogenic: a harmful insect or pathogen (optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen, wherein optionally the vector borne pathogen is: Malaria, Dengue, Yellow Fever, West Nile Virus, Zika or Chikungunya), a bacterial or fungal infection, and optionally the viral pathogen is HIV-1, Poliovirus or Hepatitis C.

8. The genetically engineered bacteria of any of claims 1 to 7, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA can kill or neutralize a pathogen in or on a: fin fish, mollusks (molluscs), or any organism in Crustacean aquaculture (optionally including: roundworms, nematodes, copepods, mites, and viruses that infect these organisms).

9. The genetically engineered bacteria of any of claims 1 to 8, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA can treat or ameliorate, or slow the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease.

10. The genetically engineered bacteria of any of claims 1 to 9, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA can treat or ameliorate, or slow the progress of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non-coding DNA targets an oncogene.

11. The genetically engineered bacteria of any of claims 1 to 10, wherein the genetically engineered bacteria is isolated from or is derived from: an animal microbiome, optionally a gut microbiome, or a human microbiome, or an insect microbiome, or a plant microbiome; or, an environmental bacteria.

12. A cell comprising or having contained therein a genetically engineered bacteria of any of claims 1 to 11.

13. The cell of claim 12, wherein the cell is a eukaryotic cell, optionally anPATENT 5810.157405PCT / Shikuma-N6 animal, a plant cell, or a human cell.

14. A method for administering or delivering a non-coding RNA or the synthetic single-stranded non-coding DNA to an organism or an individual in need thereof, comprising administering or delivering to the individual in need thereof a genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13.

15. The method of claim 14, wherein the genetically engineered bacteria or the cell is delivered or administered orally or implanted or delivered or administered rectally, or delivered or administered by inhalation, and optionally when the genetically engineered bacteria or the cell is delivered or administered orally or implanted or administered rectally or delivered or administered by inhalation the genetically engineered bacteria or the cell is formulated in an implant, a liquid, a powder, a tablet, a pill, a gel, a geltab, a lyophilate or a freeze-dried material or formulation, an aerosol or a spray, and optionally the liquid is a sterile water or saline.

16. The method of claim 14 or claim 15, wherein the genetically engineered bacteria or the cell is administered or delivered to: any animal, optionally any domesticated, farm, lab or wild animal, or to a human, a multicellular plant, any Mollusca, optionally any Porifera, Annelida, Crustacea, Echinoidea, and including oysters, clams, abalones, and mussels, optionally any Bivalvia, or any Pteriomorphia, or any animal of the group: Anomiidae (saddle oysters), Dimyidae (dimyarian oysters), Ostreidae (true oysters), Placunidae (windowpane oysters), Pteriidae (feather oysters), Spondylidae (spiny oysters), or any Anomioidea, Ostreida, Ostreoidea, Pectinoidea or Pterioidea and the like; or any Pteriomorphia (marine mussel), Palaeoheterodonta (freshwater mussel) or Heterodonta (zebra mussel); or any Atlantic Hard-Shell Clams (Mercenaria mercenaria), Soft-shell clams (Mya arenaria), Manila clams (Venerupis philippinarum), Surf clams (Spisula solida), Pacific razor clams (Siliqua patula), Atlantic Jackknife clam (Ensis leei) or Pacific Geoduck (Panopea generosa), or any abalone of the genus Haliotis.PATENT 5810.157405PCT / Shikuma-N6 any Crustacea, optionally any crab, lobster, krill, shrimp, prawn, and crayfish, including for example any animal of the clades Oligostraca, Multicrustacea, and Allotriocarida, any animal of the group: Brachyura (True Crab), Lithodidae (King crab), Pseudothelphusidae (Freshwater crabs), Potamoidea (Freshwater crabs), Portunoidea (Swimming crabs), Trapexioidea (Coral crabs), Dromioidea (Sponge crab), Decapoda (Decopods), Nephropidae (Lobster), Palinuridae (Spiny Lobster), Scyllaridae (Slipper lobster), Dendrobranchiata (Prawns), Pleocyemata (Shrimp and Crayfish), and Euphausiacea (Krill), any invertebrate coral reef organism, optionally any organism of the phylum Cnidaria, optionally any jellyfish, anemones, stony corals, soft corals, fan corals, optionally any animal of the subclasses Aurelia (moon jelly), Nematostella (starlet sea anemone), Cassiopea (upside-down jellyfish), Aiptasia (anemone), Heliporacea (crystalline octocoral), Alcyonaecea (soft coral), Scleractinia (stony corals), and Antipatharians (black coral), any Porifera, optionally any sponge classes including Demospngiae, Hexactinellida (Glass sponges), Calcarea (Calcareous sponges), or Homoscleromorpha, optionally any Xestospongia muta (Giant Barrel Sponge), Spheciospongia vesparium (Loggerhead sponge), Irchnia felix (Brown Branching sponge), Aplysinia fulva (Yellow Rope), Ircinia campana (Vase), Hippospongia lachne (Sheepswool sponge), Spongia barbara (Bath sponge), Spongia barbara dura, and Spongia graminea, any Annelida, optionally any Annelid class organism, optionally any Polychaeta, Clitellata, Machaeridia, and subphylum Sipuncula, any kelp, or any large brown algae or seaweeds that make up the order Laminariales, including Agaraceae, Akkesiphycaceae, Alariaceae, Aureophycaceae, Chordaceae, Laminariaceae, Lessoniaceae, Pseudochordaceae, also including Chlorophyta (green algae), Phaeophyceae (brown algae), Phaeothamniophyceae, Chrysophyceae (gold algae), Rhodophyta (red algae).

17. The method of any of claims 14 to 16, wherein the delivering or administering of the non-coding RNA or the synthetic single-stranded non-codingPATENT 5810.157405PCT / Shikuma-N6 DNA to the organism or the individual in need thereof, results in or causes the killing or neutralizing of a harmful insect or a pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen.

18. The method of any of claims 14 to 17, wherein the delivering or administering of the non-coding RNA or the synthetic single-stranded non-coding DNA to the individual in need thereof, results in or causes treating or ameliorating, or slowing the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is a cancer or tumor, Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease.

19. Use of genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for killing or neutralizing: a harmful insect or pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen.

20. Use of genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for treating or ameliorating, or slowing the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder is Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease.

21. Use of genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for treating or ameliorating, or slowing the progress of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non- coding DNA targets an oncogene.

22. A genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for use in killing or neutralizing: a harmful insect or pathogen, and optionally the pathogen is a viral, fungal or bacterial pathogen, or a vector borne pathogen.

23. A genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for use in treating or ameliorating, or slowing the progress of, a genetic disorder linked to a specific gene, wherein optionally the genetic disorder isPATENT 5810.157405PCT / Shikuma-N6 Huntington’s Disease, Parkinson’s Disease or Alzheimer's Disease.

24. A genetically engineered bacteria of any of claims 1 to 11, or a cell of any of claims 12 to 13, for use in treating or ameliorating, or slowing the progress of, a cancer or tumor, wherein the non-coding RNA or the synthetic single-stranded non- coding DNA targets an oncogene.

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