Production of dsRNA in several plant cells for pest control by gene silencing
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2020-03-12
- Publication Date
- 2026-04-10
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Abstract
Description
[0001] This application is a divisional application of the original application having an application date of March 12, 2020, application number 202080035482.8 (International application number PCT / IB2020 / 052245), entitled “Production of dsRNA in multiple plant cells for pest control by gene silencing”.
[0002] Related Applications This application claims priority to UK patent application No. 1903521.1, filed March 14, 2019, the contents of which are incorporated by reference in their entirety.
[0003] SEQUENCE LISTING The ASCII file, entitled 81321 Sequence Listing.txt, created on March 12, 2020, comprising 73,728 bytes, submitted with the filing of this application, is incorporated herein by reference. TECHNICAL FIELD
[0004] Some embodiments of the present invention relate to the production and amplification of multiple dsRNA molecules in a host cell for silencing of multiple pest target genes. BACKGROUND
[0005] Recent advances in genome editing technology have made it possible to change DNA sequences in living cells by editing only a few of the billions of nucleotides in the genome. Over the past decade, the tools and expertise for using genome editing have developed to the point that the methods are now widely exploited as a strategy for treating human disease. The underlying process depends on creating a site-specific DNA double-strand break (DSB) in the genome, then allowing the cell’s endogenous DSB repair machinery to repair the break, for example, by non-homologous end-joining (NHEJ) or homologous recombination (HR), the latter of which can allow precise one or more nucleotide changes to the DNA sequence using an exogenously provided donor template (Porteus, Annu Rev Pharmacol Toxicol, 2016, 56:163-90).
[0006] Three main approaches use mutagenic genome editing (NHEJ) of several cells, for example for potential therapies: (a) by creating spatially precise insertions or deletions to knock out functional genetic elements, (b) creating insertions or deletions to compensate for potential frameshift mutations; thus reactivating partly- or non-functional genes, and (c) creating definitive gene deletions. While there are several different applications using NHEJ for editing, the most widespread editing application can utilize homologous recombination (HR) for genome editing, which is highly precise as homologous recombination (HR) relies on an exogenously provided template to copy the correct sequence in the repair process, although this is a rare event.
[0007] The four main application types of HR-mediated genome editing today are: (a) gene correction (i.e., correction of diseases caused by point mutations in a single gene), (b) functional gene correction (i.e., correction of diseases caused by point mutations scattered throughout the gene), (c) safe harbor gene addition (i.e., when precise regulation is not needed or supra non-physiological levels of a transgene are needed), and (d) targeted transgene addition (i.e., when precise regulation is necessary) (Porteus, 2016, supra).
[0008] Previous work on genome editing of the RNA molecule in various eukaryotes (e.g., murine, human, shrimp, plant) has mainly focused on knocking out miRNA gene activity or altering their binding sites in target RNAs, for example: With respect to genome editing in human cells, Jiang et al. (Jiang et al., RNA Biology, 2014, 11(10): 1243-9) used CRISPR / Cas9 to remove human miR-93 from a cluster by targeting its 5' region in HeLa cells. The region targeted contained the Drosha processing site (i.e., the location in the nucleus of a host cell where Drosha, a double-stranded RNA-specific RNase III enzyme, binds, cleaves, and thereby processes primary miRNA (pri-miRNA) into pre-miRNA) and several seed sequences (i.e., conserved heptametrical sequences essential for miRNA binding to mRNA, usually located at positions 2-7 of the 5' end of the miRNA). According to Jiang et al., even a single nucleotide deletion resulted in complete knock-out of the targeted miRNA with high specificity.
[0009] With respect to genome editing in the mouse species, Zhao et al. (Zhao et al., Scientific Reports, 2014, 4:3943) provided a miRNA inhibition strategy using the CRISPR-Cas9 system in murine cells. Zhao used specially designed sgRNAs to knock out miRNAs in these cells by the Cas9 nuclease cleaving the miRNA gene at a single site.
[0010] With respect to genome editing in plants, Bortesi and Fischer (Bortesi and Fischer, Biotechnology Advances, 2015, 33:4-52) discussed the use of CRISPR-Cas9 technology in plants compared to ZFNs and TALENs, and Basak and Nithin (Basak and Nithin, Front Plant Sci., 2015, 6: 1001) taught that CRISPR-Cas9 technology has been applied to knock out protein-coding genes in several model plants such as Arabidopsis thaliana and tobacco, as well as in several crops such as wheat, maize, and rice.
[0011] In addition to disrupting miRNA activity or several target binding sites, gene silencing of several endogenous and exogenous target genes using several artificial miRNAs (amiRNAs) has also been achieved (Tiwari et al., Plant Mol Biol, 2014, 86: 1). Similar to miRNAs, amiRNAs are single-stranded, about 21 nucleotides (nt) in length, and are designed by replacing the mature miRNA sequences of a duplex in a pre-miRNA (Tiwari et al., 2014, supra). These amiRNAs are introduced as a transgene into an artificial expression cassette (including a promoter, terminator, etc.) (Carbonell et al., Plant Physiology, 2014, pp. 113.234989) and are processed and downregulate target expression by small RNA biogenesis and silencing machinery. According to Schwab et al. (Schwab et al., Plant Cell, 2006, vol. 18, 1121-1133), amiRNAs are active when expressed under tissue-specific or inducible promoters and can be used for specific gene silencing in several plants, particularly when several related but not identical target genes need to be downregulated.
[0012] Senis et al. (Senis et al., Nucleic Acids Research, 2017, vol. 45(1): e3) disclose engineering a promoterless antiviral RNAi hairpin into an endogenous miRNA locus. Specifically, Senis et al. insert an amiRNA precursor transgene (hairpin pri-amiRNA) into a location adjacent to a naturally occurring miRNA gene (e.g., miR122) by homology-directed DNA recombination induced by sequence-specific nucleases (e.g., Cas9 or TALEN nucleases). This approach uses promoterless and terminatorless amiRNAs by exploiting a transcriptionally active DNA locus that expresses a natural miRNA (miR122), i.e., the endogenous promoter and terminator drive and regulate the transcription of the inserted amiRNA transgene.
[0013] Various DNA-free methods for introducing RNA and / or several proteins into several cells have been previously described. For example, RNA transfection using electroporation and lipid transfection has been described in U.S. Patent Application No. 20160289675. Cho et al. ("Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins", Genetics, 2013, 195:1177-1180) described the direct delivery of several Cas9 / sgRNA ribonucleoprotein (RNP) complexes into several cells by microinjection of the Cas9 protein and several sgRNA complexes. Kim et al. ("Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9ribonucleoproteins", Genome Res., 2014, 24:1012-1019) described the delivery of Cas9 protein / sgRNA complexes via electroporation. Zuris et al. ("Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and invivo", Nat Biotechnol., 2014, doi: 10.1038 / nbt.3081) reported the delivery of several Cas9 protein-associated sgRNA complexes via liposomes. Summary of the Invention
[0014] According to one aspect of some embodiments of the present invention, a method is provided for generating a long dsRNA molecule in a plant cell capable of silencing a harmful organism gene, the method comprising: (a) selecting a nucleic acid sequence in a plant genome, the nucleic acid sequence encoding a silencing molecule, the silencing molecule targeting a plant gene, the silencing molecule being capable of recruiting an RNA-dependent RNA polymerase (RdRp); (b) modifying a nucleic acid sequence of the plant gene to impart silencing specificity to the harmful organism gene, such that a transcript of the plant gene including the silencing specificity forms base complementarity with the silencing molecule capable of recruiting the RdRp, thereby generating the long dsRNA molecule capable of silencing the harmful organism gene, thereby generating the long dsRNA molecule in the plant cell capable of silencing the harmful organism gene.
[0015] According to one aspect of some embodiments of the present invention, a method is provided for generating a long dsRNA molecule in a plant cell, the long dsRNA in the plant cell being capable of silencing a harmful organism gene in the plant cell, the method comprising: (a) selecting a nucleic acid sequence of a plant in a genome, the nucleic acid sequence encoding a silencing molecule, the silencing molecule targeting a plant gene, the silencing molecule being capable of recruiting RNA-dependent RNA polymerase (RdRp); (b) modifying a nucleic acid sequence of the plant gene to impart silencing specificity to the harmful organism gene, such that a transcript of the plant gene including the silencing specificity forms base complementarity with the silencing molecule capable of recruiting the RdRp, to generate the long dsRNA molecule in the plant cell, the long dsRNA molecule in the plant cell being capable of silencing the harmful organism gene in the plant cell.
[0016] According to one aspect of some embodiments of the present invention, a method is provided for generating a long dsRNA molecule in a plant cell capable of silencing a harmful organism gene, the method comprising: (a) selecting a nucleic acid sequence of a plant gene, the nucleic acid sequence of the plant gene showing a predetermined sequence homology with a nucleic acid sequence of the harmful organism gene; and (b) modifying a plant endogenous nucleic acid sequence encoding an RNA molecule to impart silencing specificity to the plant gene, such that a plurality of small RNA molecules capable of recruiting RNA-dependent RNA polymerase (RdRp) to form base complementarity with a transcript of the plant gene, the plurality of small RNA molecules being processed from the RNA molecule to generate the long dsRNA molecule capable of silencing the harmful organism gene, thereby generating the long dsRNA molecule in the plant cell capable of silencing the harmful organism gene.
[0017] According to one aspect of some embodiments of the present invention, a method for producing a pest-resistant or pest-tolerant plant is provided, the method comprising producing a long dsRNA molecule in a plant cell, the long dsRNA molecule in the plant cell being capable of silencing a pest gene according to some embodiments of the present invention.
[0018] According to one aspect of some embodiments of the present invention, a plant is provided, which is produced by the method described in some embodiments of the present invention.
[0019] According to one aspect of some embodiments of the present invention, a cell is provided, said cell being derived from the plant described in some embodiments of the present invention.
[0020] According to one aspect of some embodiments of the present invention, a seed is provided, the seed being derived from the plant described in some embodiments of the present invention.
[0021] According to one aspect of some embodiments of the present invention, a method for producing a pest-resistant or pest-tolerant plant is provided, the method comprising: (a) breeding a plant according to some embodiments of the present invention; and (b) selecting a plurality of progeny plants that express the long dsRNA molecule capable of inhibiting the pest gene and do not include the DNA editing agent, thereby producing the pest-resistant or pest-tolerant plant.
[0022] According to one aspect of some embodiments of the present invention, a method for producing a plant or plant cell according to some embodiments of the present invention is provided, the method comprising culturing the plant or plant cell under conditions that allow for reproduction.
[0023] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 21 to 24 nucleotides.
[0024] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 21 nucleotides.
[0025] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 22 nucleotides.
[0026] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 23 nucleotides.
[0027] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 24 nucleotides.
[0028] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp consists of 21 nucleotides.
[0029] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp consists of 22 nucleotides.
[0030] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp consists of 23 nucleotides.
[0031] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp consists of 24 nucleotides.
[0032] According to some embodiments of the present invention, the silencing molecules capable of recruiting the RdRp are selected from the group consisting of: trans-acting siRNA (tasiRNA), phased small interfering RNA (phasiRNA), microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), extracellular RNA (exRNA), repetitive derived RNA, and autonomous and non-autonomous transposable RNA.
[0033] According to some embodiments of the present invention, miRNA comprises a mature small RNA of 22 nucleotides.
[0034] According to some embodiments of the present invention, the miRNA is selected from: miR-156a, miR-156c, miR-162a, miR-162b, miR-167d, miR-169b, miR-173, miR -393a,miR-393b,miR-402,miR-403,miR-447a,miR-447b,miR-447c,miR-472,miR-771,miR-777,miR-828,miR-830,miR-831 ,miR-831,miR-833a,miR-833a,miR-840,miR-845b,miR-848,miR-850,miR-853,miR-855,miR-856,miR-864,miR-2933a,miR The group consisting of miR-2933b, miR-2936, miR-4221, miR-5024, miR-5629, miR-5648, miR-5996, miR-8166, miR-8167a, miR-8167b, miR-8167c, miR-87e6187d, miR-8167f, miR-8177 and miR-8182.
[0035] According to some embodiments of the present invention, the plant gene is a non-protein-coding gene.
[0036] According to some embodiments of the present invention, the plant gene is a coding gene.
[0037] According to some embodiments of the present invention, the plant gene does not encode a molecule having an intrinsic silencing activity.
[0038] According to some embodiments of the present invention, the method further includes introducing a DNA editing agent into the plant cells, the DNA editing agent conferring a silencing specificity on the plant gene against the pest gene.
[0039] According to some embodiments of the present invention, the modification of step (b) includes introducing a DNA editing agent into the plant cell, the DNA editing agent conferring a silencing specificity on the plant gene against the pest gene.
[0040] According to some embodiments of the present invention, the plant gene encodes a molecule that has an intrinsic silencing activity against a natural plant gene.
[0041] According to some embodiments of the present invention, the method further includes introducing a DNA editing agent into the plant cells, the DNA editing agent specifically redirecting a silencing of a plant gene to a harmful organism gene, the harmful organism gene being different from the natural plant gene.
[0042] According to some embodiments of the present invention, the method further includes introducing a DNA editing agent into the plant cells, the DNA editing agent specifically redirecting a silencing of a plant gene to a harmful organism gene, the harmful organism gene being different from a natural plant gene.
[0043] According to some embodiments of the present invention, the modification of step (b) includes introducing a DNA editing agent into the plant cell, the DNA editing agent specifically redirecting a silencing of the plant gene to the harmful organism gene, the harmful organism gene being different from a natural plant gene.
[0044] According to some embodiments of the present invention, the plant genes having the intrinsic silencing activity are selected from the group consisting of: trans-acting siRNA (tasiRNA), phased small interfering RNA (phasiRNA), microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), extracellular RNA (exRNA), and autonomous and non-autonomous transposable RNA.
[0045] According to some embodiments of the present invention, the plant gene having the intrinsic silencing activity encodes a phase-specific secondary siRNA producing molecule.
[0046] According to some embodiments of the present invention, the plant gene having the intrinsic silencing activity is a trans-acting siRNA (TAS) producing molecule.
[0047] According to some embodiments of the present invention, the silencing specificity of the plant gene is determined by measuring the transcriptional level of a harmful organism gene.
[0048] According to some embodiments of the present invention, the silencing specificity of the plant gene is determined phenotypically.
[0049] According to some embodiments of the present invention, the phenotypic determination is achieved by determining the plant's resistance to pests.
[0050] According to some embodiments of the present invention, the silencing specificity of the plant gene is determined by the genotype.
[0051] According to some embodiments of the present invention, a plant genotype is determined prior to a plant phenotype.
[0052] According to some embodiments of the present invention, a plant phenotype is determined prior to a plant genotype.
[0053] According to some embodiments of the present invention, the silencing specificity of the plant gene is determined by measuring the transcriptional level of the harmful organism gene.
[0054] According to some embodiments of the present invention, the determined phenotype is achieved by determining the plant's pest resistance. The determination by phenotype is achieved by determining the plant's pest resistance.
[0055] According to some embodiments of the present invention, the predetermined sequence homology includes 75% to 100% identity.
[0056] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp comprise 21 to 24 nucleotides.
[0057] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp comprise 21 nucleotides.
[0058] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp comprise 22 nucleotides.
[0059] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp comprise 23 nucleotides.
[0060] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp comprise 24 nucleotides.
[0061] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp consist of 21 nucleotides.
[0062] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp consist of 22 nucleotides.
[0063] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp consist of 23 nucleotides.
[0064] According to some embodiments of the present invention, the plurality of small RNA molecules capable of recruiting the RdRp consist of 24 nucleotides.
[0065] According to some embodiments of the present invention, the small RNA molecules capable of recruiting the RdRp are selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), trans-acting siRNA (tasiRNA), phased small interfering RNA (phasiRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), extracellular RNA (exRNA), repetitive derived RNA, and autonomous and non-autonomous transposable RNA.
[0066] According to some embodiments of the present invention, the RNA molecule does not have an intrinsic silencing activity.
[0067] According to some embodiments of the present invention, the method further includes introducing a DNA editing agent into the plant cell, the DNA editing agent conferring a silencing specificity on the RNA molecule for a plant gene.
[0068] According to some embodiments of the present invention, the RNA molecule has an intrinsic silencing activity against a natural plant gene.
[0069] According to some embodiments of the present invention, the method further includes introducing a DNA editing agent into the plant cell, the DNA editing agent redirecting the silencing of the RNA molecule to the plant gene, the plant gene being different from the natural plant gene.
[0070] According to some embodiments of the present invention, the modification in step (b) includes the introduction of a DNA editing agent into the plant cell, the DNA editing agent redirecting the silencing specificity of the RNA molecule to the plant gene, the plant gene being different from a natural plant gene.
[0071] According to some embodiments of the present invention, the plant gene that shows homology of the predetermined sequence with the nucleic acid sequence of the harmful organism gene does not encode a silencing molecule.
[0072] According to some embodiments of the present invention, the silencing specificity of the RNA molecule is determined by measuring the transcriptional level of the plant gene or the harmful organism gene.
[0073] According to some embodiments of the present invention, the silencing specificity of the RNA molecule is determined phenotypically.
[0074] According to some embodiments of the present invention, the phenotypic determination is achieved by determining the plant's resistance to pests.
[0075] According to some embodiments of the present invention, the silencing specificity of the RNA molecule is determined by genotype.
[0076] According to some embodiments of the present invention, a plant phenotype is determined prior to a plant genotype.
[0077] According to some embodiments of the present invention, a plant genotype is determined prior to a plant phenotype.
[0078] According to some embodiments of the present invention, the DNA editing agent comprises at least one sgRNA.
[0079] According to some embodiments of the present invention, the DNA editing agent comprises at least one sgRNA, said at least one sgRNA being operatively linked to a plant-expressible promoter.
[0080] According to some embodiments of the present invention, the DNA editing agent does not contain a nuclease.
[0081] According to some embodiments of the present invention, the DNA editing agent comprises a nuclease.
[0082] According to some embodiments of the present invention, the DNA editing agent is selected from a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-endonucleases, dCRISPR-endonucleases, and homing endonucleases.
[0083] According to some embodiments of the present invention, the endonuclease comprises Cas9.
[0084] According to some embodiments of the present invention, the DNA editing agent is applied to cells in the form of DNA, RNA, or RNP.
[0085] According to some embodiments of the present invention, the DNA editing agent is linked to a reporter gene for monitoring expression in a plant cell.
[0086] According to some embodiments of the present invention, the reporting molecule is a fluorescent protein.
[0087] According to some embodiments of the present invention, the plant cell is a protoplast.
[0088] According to some embodiments of the present invention, the dsRNA molecule may be processed by a cellular RNAi processing machine.
[0089] According to some embodiments of the present invention, the dsRNA molecule is processed into several secondary small RNAs.
[0090] According to some embodiments of the present invention, the dsRNA and / or the secondary small RNA contain a silencing specificity against a harmful organism gene.
[0091] According to some embodiments of the present invention, the pest is an invertebrate.
[0092] According to some embodiments of the present invention, the harmful organism is selected from a virus, an ant, a termite, a bee, a wasp, a caterpillar, a cricket, a locust, a beetle, a snail, a slug, a nematode, a bedbug, a fly, a fruit fly, a whitefly, etc. A group consisting of a hitefly, a mosquito, a grasshopper, a planthopper, an earwig, an aphid, a scale, a thrips, a spider, a mite, a psyllid, a tick, a moth, a worm, a scorpion, and a fungus.
[0093] According to some embodiments of the invention, the plant is selected from a group consisting of a crop, a flowering plant, a weed, and a tree.
[0094] According to some embodiments of the present invention, the plant is non-GMO.
[0095] According to some embodiments of the present invention, the plant is a genetically modified plant.
[0096] According to some embodiments of the present invention, the plant is non-genetically modified (non-GMO).
[0097] According to some embodiments of the present invention, the plant is genetically modified (GMO).
[0098] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although several methods and materials similar to or equivalent to those described herein may be used in the practice or testing of several examples of this invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to impose any necessary limitations. Attached Figure Description
[0099] Some embodiments of the invention are illustrated herein by way of example only and with reference to the accompanying drawings. Reference will now be made specifically to the drawings, with emphasis placed on the details shown by way of example and for the purpose of an illustrative discussion of several embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make it clear to those skilled in the art how embodiments of the invention can be practiced.
[0100] In the aforementioned figures: Figure 1 This is a photograph illustrating the first proposed model (referred to as Model 1) for target gene amplification via gene editing-induced gene silencing (GEiGS). According to this model (see several corresponding numbers in several diagrams): 1. The harmful organism gene "X" is the target gene (when silenced, the harmful organism is controlled).
[0101] 2. Identify the host-related gene - X (plant gene "X") through homology search.
[0102] 3. Perform GEiGS to redirect the silencing specificity of an amplifier small RNA (e.g., 22nt miRNAs) targeting plant gene “X”.
[0103] 4. The amplifier small GEiGS RNA forms a RISC complex associated with RdRp (the amplification enzyme).
[0104] 5. RdRp synthesizes an antisense RNA chain that is complementary to the transcript of plant gene "X", forming dsRNA.
[0105] 6. The plant gene “X” dsRNA is processed into secondary sRNA by (several) dicers or similar proteins.
[0106] 7. The plant gene "X" dsRNA is absorbed by several harmful organisms. Among these harmful organisms, the plant dsRNA-X is processed into small RNA, which regulates the corresponding homologous harmful organism gene "X" through RNAi.
[0107] 8. Possibly, several secondary sRNAs are absorbed by several harmful organisms and silence the target gene "X".
[0108] Figure 2This is a photograph illustrating the second proposed model (referred to as Model 2) for target gene amplification via GEiGS. According to this model (see several corresponding numbers in several diagrams): 1. The harmful organism gene "X" is the target gene (when silenced, the harmful organism is controlled).
[0109] 2. Perform GEiGS to redirect the naturally occurring amplified RNAi precursor silencing specificity (e.g., TAS; amplified and processed into several tasiRNAs) targeting the harmful organism gene “X”.
[0110] 3. A wild-type amplifier sRNA forms a RISC complex associated with RdRp (amplifier enzyme).
[0111] 4. The RdRp synthesizes an antisense RNA strand that is complementary to the transcript of the amplified GEiGS precursor, thereby forming dsRNA.
[0112] 5. The amplified GEiGS dsRNA is processed into several secondary sRNAs by dicer(s).
[0113] 6. GEiGS dsRNA is absorbed by the harmful organism. In the harmful organism, the plant GEiGS-dsRNA is processed into small RNA, which regulates the corresponding homologous harmful organism gene "X" through RNAi.
[0114] 7. Possibly, secondary sRNAs from GEiGS-dsRNA (e.g., tasiRNA in TAS precursors) may also be absorbed by the harmful organism and silence the target gene "X".
[0115] Figure 3A This describes the identification of several endogenous genes in the plant that have several regions homologous to the sequences of the pest (each model 1). Specifically, it describes the alignment of the AF502391.1 (soybean cyst nematode (H. glycines), SEQ ID NO: 1) pest with the NM_001037071.1 (Arabidopsis thaliana, SEQ ID NO: 2) plant gene.
[0116] Figure 3B This describes GEiGS oligonucleotides based on miRNA, designed to carry several siRNA sequences targeting a downstream region of the homologous region in the plant. Figure 3A(As described in the text). Top: GEiGS oligonucleotide, SEQ ID NO: 3 (siRNA shown in red). Bottom: Plant target gene homologous to the pest (SEQ ID NO: 4). Homologous pest sequence (SEQ ID NO: 1) is shown in green. The sequence predicted as targeted by GEiGS-siRNA is shown in red.
[0117] Figure 4A This describes the identification of several endogenous genes in the plant that have several regions homologous to the sequences of the pest (each model 1). Specifically, the comparison of the AF500024.1 (soybean cyst nematode, SEQ ID NO: 5) pest gene with the NM_116351.7 (Arabidopsis thaliana, SEQ ID NO: 6) plant gene.
[0118] Figure 4B This describes GEiGS oligonucleotides based on miRNA, designed to carry several siRNA sequences targeting a downstream region of the homologous region in the plant. Figure 4A (As described in the text). Top: GEiGS oligonucleotide, SEQ ID NO: 7 (siRNA shown in red). Bottom: Target gene homologous to the said pest (SEQ ID NO: 8). Homologous pest sequence (SEQ ID NO: 5) shown in green. The sequence predicted as targeted by GEiGS-siRNA is shown in red.
[0119] Figure 5A This describes the identification of several endogenous genes in the plant that have several regions homologous to the sequences of the pest (each model 1). Specifically, the comparison of the AF469060.1 (soybean cyst nematode, SEQ ID NO: 9) pest genes with those of the NM_001203752.2 (Arabidopsis thaliana, SEQ ID NO: 10) plant.
[0120] Figure 5B This describes GEiGS oligonucleotides based on miRNA, designed to carry several siRNA sequences targeting a downstream region of the homologous region in the plant. Figure 5A (As described in the text). Top: GEiGS oligonucleotide, SEQ ID NO: 11 (siRNA shown in red). Bottom: Target gene homologous to the said pest (SEQ ID NO: 12). Homologous pest sequence (SEQ ID NO: 9) shown in green. The sequence predicted as targeted by GEiGS-siRNA is shown in red.
[0121] Figure 6This is a flowchart of one embodiment of a computational pipeline that generates several GEiGS templates. The computational GeiGS pipeline applies biological metadata and can automatically generate several GeiGS DNA donor templates for minimally editing several endogenous non-coding RNA genes (e.g., miRNA genes) to obtain a new functional gain, namely, redirecting their silencing ability to the expression of target genes of interest.
[0122] Figure 7 This is a flowchart of an embodiment of Genome Editing Induced Gene Silencing (GEiGS) using siRNA targeting the PDS gene to replace endogenous miRNA, thereby inducing gene silencing of the endogenous PDS gene. To introduce this modification, a two-component system is used. First, a CRISPR / CAS9 system in a GFP-containing vector generates a cut at a selected locus via several designed specific guide RNAs to promote homologous DNA repair (HDR) at that site. Second, a donor sequence (with the required modification of the miRNA sequence) is introduced as a template for the HDR to target several newly assigned genes. This system is being used in protoplast transformation, where it is recovered and regenerated into several plants via FACS enrichment (due to the GFP signal in the CRISPR / CAS9 vector).
[0123] Figures 8A-8C These are several photographs illustrating photobleaching caused by the silencing of the PDS gene. (Tobacco (Nicotiana) (Figures 8A to 8B)) Figure 8B The silencing of the PDS gene in *Nicotiana benthamiana* (Fig. 8B) and *Arabidopsis* (Fig. 8C) caused photobleaching in these plants. The photographs were taken 3.5 weeks after PDS silencing.
[0124] Figure 9AThis diagram illustrates an example of several HDR-mediated genomic swaps in several Col-0 cells and several primers used for PCR and genotyping of such swaps. The CRISPR / Cas9 and sgRNA target the swapped regions, resulting in a dsDNA break. Several donor templates carry several homologous arms for insertion into the genomic sites (AtTAS1b or AtTAS3a) via homology-directed repair (HDR), thereby introducing the desired swaps. Swap region: modified to target several nematode gene sequences. Several short arrows represent swap-specific or wt-specific forward primers and non-specific reverse primers, applicable to all reactions for PCR to illustrate the genomic swaps. The reverse primers are designed to be further annealed downstream of the recombination site to avoid amplification of the donor template. Several exchange-specific forward primers were designed to allow amplification only when an exchange occurs. An additional forward primer was designed for control PCR amplification of wild-type (WT) sequences only. Dashed lines represent PCR products. Ellipses represent the reverse primers used in the Sanger sequencing reaction.
[0125] Figures 9B-9C Several electrophoretic micrographs of several PCR products produced using several WT primers are shown. The nonspecific reverse primer and one WT-specific primer were used to perform PCR on DNA extracted from all the treatments described in Example 3. Several PCR products were run on a 1.6% agarose gel. Several small arrows and numbers indicate several expected bands and sizes of the several PCR products. Figure 9B represents several PCR reactions at the AtTAS1b locus, and... Figure 9C Several reactions representing the AtTAS3a locus. Y25: Y25, the β subunit of the COPI complex; Splicing: splicing factor; Ribo3a: ribosomal protein 3a; Spliceo: spliceosome SR protein; WT: wild type; H2O: template-free, water-negative PCR control group; MW: 1 kb plus molecular weight ladder (NEB).
[0126] Figures 9D-9ESeveral electrophoretic micrographs of several PCR products produced using several exchange-specific primers are shown. The non-specific reverse primer and one exchange-specific forward primer were used to perform PCR on DNA extracted from all exchange treatments in Example 3. WT DNA was also used as a template as a control for the specificity of the reaction. Several PCR products were run on a 1.6% agarose gel. Several small arrows and numbers indicate the expected bands and sizes of the several PCR products. Figure 9D represents several PCR reactions at the AtTAS1b (Tas1b) locus exchange, and Figure 9E represents several reactions at the AtTAS3a (Tas3a) locus exchange. Y25: Y25, β subunit of the COPI complex; Splicing: splicing factor; Ribo3a: ribosomal protein 3a; Spliceo: spliceosome SR protein; WT: wild type; H2O: template-free, water-negative PCR control; MW: 1 kb plus molecular weight ladder (NEB).
[0127] Figures 9F-9G A scheme for a Sanger sequencing reaction of several PCR products is illustrated. The nonspecific reverse primers shown in Figure 9A are used for Sanger sequencing of each PCR product. Several arrows represent the specific forward primers used for PCR amplification. Several other nucleotide changes introduced after the HDR event (not originating from the primers used in the reaction) are highlighted and shown in gray. Several chromatograms show the sequences of the several PCR products, aligned to the several predicted sequences (upper line). Figure 9F represents several sequencing reactions with several exchanges at the AtTAS1b (Tas1b) locus, and Figure 9G represents several reactions with several exchanges at the AtTAS3a (Tas3a) locus. Y25: Y25, β subunit of the COPI complex; Splicing: splicing factor; Ribo3a: ribosomal protein 3a; Spliceo: spliceosome SR protein; WT: wild type.
[0128] Figures 10A-10B A meaningful ( ) diagram of dsRNAs generated by several genome exchanges mediated by HDR in several Col-0 cells. Figure 10A ) and antonyms ( Figure 10BSeveral schematic diagrams of the chain. Exchange region: A sequence modified to target several nematode genes. Several short arrows represent the several non-specific primers used for reverse transcription PCR (RT-PCR) and cDNA production. Several additional short arrows represent the exchange-specific and non-specific primers, common in all reactions, used for cDNA PCR (PCR) to demonstrate exchange expression. Several PCR reactions were designed such that all PCR products were less than 200 nucleotides in length. Several specific primers were designed such that they only allow amplification when exchange occurs. The several dashed lines represent the expected several PCR products. The ellipses represent the several primers used for several Sanger sequencing reactions. The directions indicate that several transcripts are from 5' to 3'.
[0129] Figures 10C-10D Several electrophoretic images are shown, illustrating the expression of several sense and antisense RNA strands of AtTAS1b to detect the expression of several PCR products containing several exchanged dsRNAs. Several RT-PCR reactions were performed to generate cDNA, and... Figures 10A-10B Several primers described herein were used in several subsequent PCR reactions. Several PCR products were run on a 1.6% agarose gel. Several small arrows and several numbers indicate several expected bands and several sizes of the several PCR products. Figure 10C represents several PCR reactions of AtTAS1b sense RNA transcripts, and Figure 10D represents PCR reactions of AtTAS1b antisense RNA transcripts. Y25: Y25, β subunit of the COPI complex; WT: wild type; H2O: template-free, water-negative PCR control; MW: 1 kb plus molecular weight ladder (NEB). +RT: Several PCR reactions using cDNA amplified by reverse transcriptase as a template. -RT: Several reverse transcription controls - no reverse transcriptase was used and no cDNA was produced.
[0130] Figures 10E-10F Several electrophoretic images are shown, illustrating the expression of several sense and antisense RNA strands of AtTAS3a to detect the expression of several PCR products containing several exchanged dsRNAs. Several RT-PCR reactions were performed to generate cDNA, and... Figures 10A-10BSeveral primers described herein were used in several subsequent PCR reactions. Several PCR products were run on a 1.6% agarose gel. Several small arrows and numbers indicate the expected bands and sizes of the several PCR products. Figure 10E represents several PCR reactions of the AtTAS3a sense RNA transcript, and Figure 10F represents several PCR reactions of the AtTAS3a antisense RNA transcript. Ribo3a: ribosomal protein 3a; WT: wild type. H2O: template-free, water-negative PCR control. MW: 1 kb plus molecular weight ladder (NEB). +RT: Several PCR reactions using cDNA amplified by reverse transcriptase as a template. -RT: Several reverse transcription controls – no reverse transcriptase was used and no cDNA was produced.
[0131] Figure 10G A scheme for a Sanger sequencing reaction of several PCR products is illustrated, which amplifies the sense strand of RNA through several introduced crossovers. The nonspecific forward primers in Figure 10A are used for Sanger sequencing of each PCR product. Several arrows represent the specific reverse primers used for PCR amplification. Several other nucleotide changes introduced by the donor template are highlighted and indicated in gray. Several chromatograms show the sequences of the several PCR products, which are aligned with the several predicted sequences. The upper figure represents several sequencing reactions demonstrating the expression of crossovers at the AtTAS1b (Tas1b) locus, and the lower figure represents a reaction demonstrating the expression of crossovers at the AtTAS3a (Tas3a) locus. Y25: Y25, β subunit of the COPI complex; Ribo3a: ribosomal protein 3a; WT: wild type.
[0132] Figure 10H A scheme for a Sanger sequencing reaction of PCR products is illustrated, which amplifies the antisense strand of RNA by introducing several crossovers. The nonspecific reverse primers in Figure 10B are used for Sanger sequencing of each PCR product. Several arrows represent the specific forward primers used for PCR amplification. Several other nucleotide changes introduced by the donor template are highlighted and indicated in gray. Several chromatograms show the sequences of several PCR products, which are aligned with several predicted sequences. The upper figure represents several sequencing reactions demonstrating the expression of crossovers at the AtTAS1b (Tas1b) locus, and the lower figure represents a reaction demonstrating the expression of crossovers at the AtTAS3a (Tas3a) locus. Y25: Y25, β subunit of the COPI complex; Ribo3a: ribosomal protein 3a; WT: wild type.
[0133] Figure 10IA scheme for a Sanger sequencing reaction amplifying several PCR products of the sense and antisense strands of wild-type RNA transcribed from Tas1b and Tas3a is illustrated. For the sense transcripts, the nonspecific forward primers from Figure 10A are used for Sanger sequencing of each PCR product. For the antisense transcripts, the nonspecific reverse primers from Figure 10B are used for Sanger sequencing of each PCR product. Several arrows represent the several forward primers used for PCR amplification. Several chromatograms show the several sequences of the several PCR products, which are aligned with the several annotated WT sequences.
[0134] Figure 11A A bar graph is provided in the figure below to illustrate several levels of TuMV infection in several leaves of *N. benthamiana* after inoculation with various treatments, as represented by quantification of several TuMV transcript levels and GFP visualization of relative expression. Controls and several treatments were infiltrated side-by-side simultaneously on the same leaf. From left to right - (1) Infiltrating the leaf using *Agrobacterium* containing a TuMV vector (n=3; left side of the leaf) or *Agrobacterium* without any vector (n=3; right side of the leaf). (2) Infiltrating the leaf using *Agrobacterium* containing a vector overexpressing miR173 (n=3; left side) or *Agrobacterium* without a vector (n=3; right side). (3) Infiltrating the leaf using a vector overexpressing GEiGS-virtual (n=3; left side) or GEiGS-TuMV (n=3; right side). (4) Agrobacterium tumefaciens containing a vector overexpressing GEiGS-virtual (n=3; left) or Agrobacterium tumefaciens containing a vector encoding the GEiGS-TuMV (n=2; right) were used to infiltrate the leaves, both in conjunction with Agrobacterium tumefaciens containing a vector overexpressing miR173. The micrographs in the above figure are representative images of several samples analyzed. TuMV was monitored by GFP signaling and visualized under UV light. Several bars represent several means; several error bars represent standard errors; *- p-value < 0.05; **- p-value < 0.01 based on one-way ANOVA and post-hoc Tukey HSD detection.
[0135] Figure 11BSeveral photographs are provided illustrating whole leaves of several *Nicotiana benthamiana* plants, in which *Agrobacterium* strains containing several vectors overexpressing GEiGS-TuMV and miR173 (middle) or GEiGS-TuMV and miR173 (right) were co-infiltrated into the whole leaves of these plants. Vector-free *Agrobacterium* was used to infiltrate control leaves (left). TuMV was monitored by GFP signaling and is visualized under UV light.
[0136] Figure 12A This is a bar chart showing the relative expression of ribosomal protein 3a in several nematodes fed with total RNA extracted from several leaves of *Nicotiana benthamiana*, with vectors modified to target ribosomal protein 3a and overexpressing miR390 and TAS3a co-infiltrated into the leaves. Several nematodes fed with RNA from explants overexpressing the TAS3a wt backbone and miR390 amplifier served as controls. Actin was used as an endogenous normalizer gene, and qRT-PCR was used to analyze several nematodes fed with RNA extracts over 3 days. (The error bars represent standard error; ***-p < 0.001).
[0137] Figure 12B This is a bar chart showing the relative expression of spliceosome SR protein in several nematodes fed with total RNA extracted from leaves of *Nicotiana benthamiana*, with vectors modified to target the spliceosome SR protein and overexpressing miR390 and TAS3a co-infiltrated into the leaves. Several nematodes fed with RNA from explants overexpressing the TAS3a wt backbone and miR390 amplifier served as controls. Actin was used as an endogenous standard gene, and qRT-PCR was used to analyze several nematodes fed with RNA extracts over 3 days. (Several error bars represent standard error; **- p < 0.01).
[0138] Figures 13A-13D The diagram illustrates the use of expression targeting ribosomal protein 3a ( Figure 13A and Figure 13B ) and spliceosome protein 3a ( Figure 13C and Figure 13D RNA sequence analysis (RNA-seq analysis) of several vectors designed by GEiGS and infiltrated into several leaves of *Nicotiana benthamiana*. Figure 13A and Figure 13C ) and small RNA sequence analysis (small RNA-seq analysis) Figure 13B and Figure 13DThe miR390, compared to the GEiGS design, infiltrates at 48 to 72 hours. The light gray rectangles in each figure represent the miR390 binding region on the transcript. The black squares in each figure represent the homologous regions for the target genes, which produce the secondary siRNAs targeting several genes in several nematodes. The top chromatograms in each figure represent the sense strand, while the bottom chromatograms represent the antisense strand. Detailed Implementation
[0139] In some embodiments of the present invention, the production and amplification of dsRNA molecules in a host cell are involved to silence several harmful biological target genes.
[0140] The principles and operation of this invention can be better understood by referring to the accompanying drawings and descriptions.
[0141] Before explaining at least one embodiment of the present invention in detail, it should be understood that the application of the present invention is not necessarily limited to the details set forth in the following description or illustrated by the various examples. The present invention can have other embodiments, or can be practiced or performed in various ways. Furthermore, it should be understood that the wording and terminology used herein are for illustrative purposes and should not be considered restrictive.
[0142] Previous efforts to edit the genomes of multiple RNA molecules in various organisms (e.g., rodents, humans, plants) primarily involved using transgenes to disrupt miRNA activity or multiple target binding sites. Genome editing in several plants has mainly focused on using CRISPR-Cas9 technology, multiple ZFNs, and multiple TALENs, among other nucleases, to knock out multiple genes or insert multiple genes in several model plants. Furthermore, gene silencing in plants using artificial miRNA transgenes to silence endogenous and exogenous target genes has been described (Molnar A et al., Plant J., 2009, 58(1):165-74. doi:10.1111 / j.1365-313X.2008.03767.x, e-version January 19, 2009; Borges and Martienssen, Nature Reviews Molecular Cell Biology (AOP), online publication November 4, 2015; doi:10.1038 / nrm4085). Several artificial miRNA transgenes were introduced into several plant cells within an artificial expression cassette (including a promoter, terminator, selection marker, etc.) to downregulate target expression.
[0143] Recent advances in several genome editing technologies have made it possible to alter the DNA sequence in several living cells by editing one or more nucleotides at several desired locations in the genome following site-specific double-strand breaks (DSBs) in human patient cells (e.g., through genome editing (NHEJ and HR)). While NHEJ is primarily (if not excluded) used for knockout purposes, HR is used to introduce precise edits at several specific sites, such as several point mutations or to correct several naturally occurring or genetically propagated harmful mutations.
[0144] Several mature small RNAs (i.e., several dicer products and several non-dicer products) and dsRNAs (i.e., several dicer substrates, such as several small RNA precursors) can mediate effective cellular gene knockdown. The biogenesis of several miRNAs involves the presence of several dsRNA structures (e.g., hairpin precursors). However, these hairpin RNAs may not be efficiently absorbed by several harmful organisms because: (i) their quantity is low due to their instability (e.g., processing by a dicing machine); and (ii) they lack the RNA-RNA amplification phase of several RNA-dependent RNA polymerases (RdRp). Therefore, several harmful organisms are more susceptible to the effects of ingested small RNA precursors (e.g., dsRNAs).
[0145] While putting this invention into practice, the inventors have designed a gene-editing technique for generating several long dsRNA molecules in several plant cells and tissues to target genes of several harmful organisms. These dsRNA molecules can move and transfer between the cells and tissues; therefore, once generated in several cells, they occur outside those cells. Furthermore, these dsRNA molecules can be transferred between several organisms by ingesting material derived from a dsRNA-expressing host (e.g., several plant leaves and stems). Specifically, several inventors have developed the GEiGS system involving one of two models.
[0146] The several models described below are in part based on the genome editing-induced gene silencing (GEiGS) technology described in WO2019 / 058255, which is incorporated herein by reference in its entirety. As used herein, the phrase “performing GEiGS” refers to using the GEiGS technology to redirect the silencing specificity of a silencing RNA, primarily involving modifying a nucleic acid sequence encoding a silencing RNA such that the encoded silencing RNA targets a selected target. According to some embodiments, GEiGS is performed by inducing a double-strand break in the nucleic acid sequence encoding the silencing RNA in a cell (e.g., by expressing or introducing a nuclease into the cell, such as, but not limited to, Cas9), and a nucleic acid template is provided comprising several desired nucleotide alterations in the nucleic acid sequence encoding the silencing RNA. According to these embodiments, the several nucleotide alterations are then introduced into the nucleic acid sequence encoding the silencing RNA by homology-dependent recombination (HDR) when the relevant portion of the nucleic acid template is introduced. According to some embodiments, the nucleic acid template introduces several nucleotide changes in the nucleic acid sequence encoding the silenced RNA, such that the silenced RNA targets a selected target sequence. Several examples of using GEiGS to modify several nucleotides in a nucleic acid sequence encoding a miRNA or a tasiRNA are illustrated in Examples 1B and 3 below.
[0147] In the first model, a plant gene homologous to a target gene of a pest is identified. GEiGS is performed to redirect the silencing specificity of a small RNA molecule to the plant gene (homogeneous to the target gene of the pest). This small RNA molecule (also called an amplifier or primer small RNA) forms a complex with RdRp, and RdRp synthesizes a complementary antisense RNA strand for the transcript of the plant gene, forming a dsRNA. The dsRNA is then further processed into several secondary small RNAs (sRNAs). Importantly, the primary small RNAs, dsRNAs, and secondary small RNA molecules (i.e., the RNAi processing products of the newly generated dsRNAs) are absorbed by the pest, for example via a Dicer-like process, and can mediate pest gene silencing. Essentially, by redirecting the targeting specificity of an amplifier small RNA molecule using GEiGS, the first model is able to generate a new long dsRNA from a sequence from which a long dsRNA was not previously formed, thereby creating a specific RNA generation site. Because the locus has a natural similarity to a pest gene, the generated long dsRNA has the ability to silence the corresponding gene within the pest.
[0148] In the second model, GEiGS is performed on a plant gene that is naturally converted into a double-stranded RNA form (producing a long dsRNA and several phased RNAs at a naturally amplified locus, such as a naturally occurring TAS), to specifically redirect silencing to a harmful target gene. Initially, a naturally silencing RNA molecule (also referred to herein as an amplifier or primer small RNA; for example, a 22nt miRNA, such as miR-173) is selected to target the plant gene and is able to form a complex with RdRp. RdRp synthesizes a complementary antisense RNA strand corresponding to the transcript of the plant gene, forming a long dsRNA. The long dsRNA is then further processed into several secondary sRNAs (i.e., the RNAi products of the newly generated dsRNAs, such as Dicer-like molecules). According to the model, the long dsRNA and the several secondary small RNA molecules are absorbed by the harmful organism and can mediate the silencing of the harmful gene.
[0149] Therefore, this invention provides the formation of amplifiable dsRNA molecules in several plant cells and tissues, with a larger expected quantity and a larger small RNA population, and thus higher silencing efficacy. Furthermore, the multiple secondary small RNAs generated from the dsRNA molecules increase the opportunities for effective target knockdown. The dsRNA molecules generated by this method are effectively absorbed by several harmful organisms, thereby enabling the effective silencing and safe control of several harmful organism genes without harming the plants. Moreover, the gene editing technology described herein does not utilize the aforementioned classical molecular genetic and transgenic tools, which include expression cassettes with a promoter, terminator, and selection marker.
[0150] Therefore, according to one aspect of the present invention, a method is provided for producing a long dsRNA molecule in a plant cell capable of silencing a harmful organism gene, the method comprising: (a) Select a nucleic acid sequence of a plant gene, wherein the nucleic acid sequence of the plant gene has a predetermined sequence homology with a nucleic acid sequence of the harmful organism gene; (b) Modifying a plant endogenous nucleic acid sequence encoding an RNA molecule to impart silencing specificity to the plant gene, such that several small RNA molecules capable of recruiting RNA-dependent RNA polymerase (RdRp) form base complementarity with a transcript of the plant gene, wherein the several small RNA molecules are processed from the RNA molecule to produce the long dsRNA molecule capable of silencing the harmful organism gene. This results in the production of long dsRNA molecules in the plant cells capable of silencing the genes of the harmful organism.
[0151] As used herein, the term "long dsRNA molecule" refers to several double-stranded sequences of a number of polynucleotides, having a first strand (sense strand) and a second strand (antisense strand) that is an antisense complement of the first strand. The polynucleotides are held together by base pairing (e.g., two sequences that are antisense complements each other in the base pairing region). The double-stranded polynucleotides may be a matrix of an enzyme from the Dicer family. Typically, the long dsRNA molecule is at least 26 bp or longer. The two strands may have the same length or different lengths, provided that there is sufficient sequence homology between the two strands to form a stable double-stranded structure, wherein at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementarity over the entire length.
[0152] The terms “complementation,” “complementarity,” or “complementary” are used to describe the hybridization of an index of RNA molecules (or at least a portion of several RNA molecules existing as processed small RNA, or at least one strand or a portion thereof of a double-stranded polynucleotide, or a portion thereof of a single-stranded polynucleotide) with a target RNA (e.g., a transcript of the plant gene) or a fragment thereof under physiological conditions to achieve RdRp-mediated regulation or function of target gene synthesis. For example, in some embodiments, when the target RNA (or several family members of a given target gene) contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, ... When compared to a sequence of 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500 or more consecutive nucleotides, an RNA molecule has 100% sequence identity or at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
[0153] As used herein, a small RNA molecule or its processed form of small RNA is said to exhibit “perfect complementarity” when each nucleotide of one of the sequences read from 5' to 3' is complementary to each nucleotide of another sequence read from 3' to 5'. A nucleotide sequence that is perfectly complementary to a reference nucleotide sequence will exhibit the same sequence as the inverse complementary sequence of the reference nucleotide sequence.
[0154] Methods for determining sequence complementarity are well known in the art, including but not limited to several bioinformatics tools well known in the art (e.g., BLAST, multiple sequence alignment).
[0155] According to one embodiment, the long dsRNA molecule is longer than 20 bp.
[0156] According to one embodiment, the long dsRNA molecule is longer than 21 bp.
[0157] According to one embodiment, the long dsRNA molecule is longer than 22 bp.
[0158] According to one embodiment, the long dsRNA molecule is longer than 23 bp.
[0159] According to one embodiment, the long dsRNA molecule is longer than 24 bp.
[0160] According to one embodiment, the long dsRNA molecule comprises 20 to 100,000 bp.
[0161] According to one embodiment, the long dsRNA molecule comprises 20 to 10,000 bp.
[0162] According to one embodiment, the long dsRNA molecule comprises 20 to 1,000 bp.
[0163] According to one embodiment, the long dsRNA molecule comprises 20 to 500 bp.
[0164] According to one embodiment, the long dsRNA molecule comprises 20 to 50 bp.
[0165] According to one embodiment, the long dsRNA molecule comprises 200 to 5000 bp.
[0166] According to one embodiment, the long dsRNA molecule comprises 200 to 1000 bp.
[0167] According to one embodiment, the long dsRNA molecule comprises 200 to 500 bp.
[0168] According to one embodiment, the long dsRNA molecule comprises 2000 to 100,000 bp.
[0169] According to one embodiment, the long dsRNA molecule comprises 2000 to 10,000 bp.
[0170] According to one embodiment, the long dsRNA molecule comprises 2000 to 5000 bp.
[0171] According to one embodiment, the long dsRNA molecule comprises 10,000 to 100,000 bp.
[0172] According to one embodiment, the long dsRNA molecule comprises 1,000 to 10,000 bp.
[0173] According to one embodiment, the long dsRNA molecule comprises 100 to 10,000 bp.
[0174] According to one embodiment, the long dsRNA molecule comprises 100 to 1,000 bp.
[0175] According to one embodiment, the long dsRNA molecule comprises 10 to 1,000 bp.
[0176] According to one embodiment, the long dsRNA molecule comprises 10 to 100 bp.
[0177] According to one embodiment, the long dsRNA molecule includes a protruding end, which is a non-double-stranded region of a dsRNA molecule (i.e., single-stranded RNA).
[0178] According to one embodiment, the long dsRNA molecule does not include a protruding end.
[0179] According to one embodiment, the long dsRNA molecule of the present invention can be processed into several small RNA molecules capable of binding to the RNA-induced silencing complex (RISC). Therefore, the long dsRNA molecule of the present invention can serve as a substrate (i.e., a precursor RNA molecule) for intracellular RNAi processing mechanisms and can be processed by ribonucleases, including but not limited to the DICER protein family (e.g., DCR1 and DCR2), the DICER-like protein family (e.g., DCL1, DCL2, DCL3, DCL4), the ARGONAUTE protein family (e.g., AGO1, AGO2, AGO3, AGO4), tRNA cleaving enzymes (e.g., RNY1, ANGIOGENIN, RNase P, RNase P-like, SLFN3, ELAC1, and ELAC2), and Piwi-interacting RNA (piRNA)-related proteins (e.g., AGO3, AUBERGINE, HIWI, HIWI2, HIWI3, PIWI, ALG1, and ALG2), into several small RNA molecules, as discussed in detail below.
[0180] As used herein, the term "plant" includes several whole plants, a grafted plant, ancestors and offspring of said several plants, and several plant parts, including several seeds, several seedlings, several stems, several roots (including several tubers), several rhizomes, several scions, and several plant cells, tissues, and organs. The plant can be in any form, including several suspension cultures, several embryos, several meristems, several callus tissues, several leaves, several gametophytes, several sporophytes, several pollen grains, and several microspores. Several plants that may be useful in the methods described in this invention include all plants belonging to the superfamily Viridiplantee, particularly monocotyledonous and dicotyledonous plants, including fodder or legumes, ornamental plants, food crops, trees or shrubs, selected from the following genera: Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp., Areca catechu, Astella fragrans, Astragalus membranaceus. cicer), Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp., Camellia sinensis, Canna indica, Cannabis, Cannabis sativa, Hemp, Industrial Hemp, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp.Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa), Dibeteropogonamplectens, Dioclea spp., Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi / losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp., Freycinetia The genera *bankli*, *Geranium thunbergii*, *Ginkgo biloba*, *Glycine javanica*, *Gliricidia spp.*, *Gossypium hirsutum*, *Grevillea spp.*, *Guibourtia coleosperma*, and *Hedysarum spp.* are mentioned.), Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia *Glyptostroboides*, *Musa sapientum*, *Banana*, *Nicotianum* spp., *Onobrychis* spp., *Ornithopus* spp., *Oryza* spp., *Peltophorum africanum*, *Pennisetum* spp., *Persea gratissima*, *Petunia* spp., *Phaseolus* spp., *Phoenix canariensis*, *Phormium cookianum*, *Photinia* spp., *Picea glauca*, *Pinus* spp., *Pisum sativam*, *Podocarpus totara*, *Pogonarthria* *Fleckii*, *Pogonaffhria squarrosa*, and *Populus* spp.), Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosas spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoia dendron The genera *Giants giganteum*, *Sorghum bicolor*, *Spinacia*, *Sporobolus fimbriatus*, *Stiburusalopecuroides*, *Stylosanthos humilis*, *Tadehagi*, *Taxodium distichum*, *Themeda triandra*, *Trifolium*, *Triticum*, *Tsuga heterophylla*, *Vaccinium*, and *Vicia*.The following vegetables and plants may be included: grapes (Vitis vinifera), Watson's cone flower (Watsonia pyramidata), calla lily (Zantedeschia aethiopica), corn (Zea mays), amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower, celery, collard greens, flax, kale, lentils, oilseed rape, okra, onions, potatoes, rice, soybeans, straw, beets, sugarcane, sunflowers, tomatoes, squash tea, and trees. Alternatively, algae and other non-viridiplantae may be used in some embodiments of the methods of this invention.
[0181] According to one specific embodiment, the plant is a crop, a flowering plant, a weed, or a bridge tree.
[0182] According to a specific embodiment, the plant is a woody plant species, such as different species of kiwifruit (Actinidia chinensis) (Actiniaceae), cassava (Manihotesculenta) (Euphorbiaceae), tulip tree (Firiodendron tulipifera) (Magnoliaceae), poplar (Populus) (Salicaceae), sandalwood (Santalaceae), elm (Ulmus) (Ulmaceae), as well as different species of Rosaceae (apple, plum, pear) and Rutaceae (citrus, lemon), gymnosperms (e.g., white spruce (Picea)). Trees (e.g., Betulaceae, Fagaceae, gymnosperms, and tropical trees), fruit trees, shrubs or herbaceous plants (e.g., bananas, cocoa, coconuts, coffee, dates, grapes, and tea), and oil palm.
[0183] According to a specific embodiment, the plant is a tropical crop, such as coffee, macadamia nuts, bananas, pineapples, taro, papayas, mangoes, barley, beans, cassava, chickpeas, cocoa, cowpeas, maize, millet, rice, sorghum, sugarcane, sweet potatoes, tobacco, taro, tea, and yams.
[0184] “Grain,” “seed,” or “bean” refers to the reproductive unit of a flowering plant that can develop into another such plant. As used herein, the terms are used synonymously and interchangeably.
[0185] According to one specific embodiment, the plant is a plant cell, such as a plant cell in an embryonic cell suspension.
[0186] According to one specific embodiment, the plant cell is a protoplast.
[0187] The protoplasts are derived from any plant tissue, such as fruit, flowers, roots, leaves, embryos, embryo cell suspensions, callus, or seedling tissue.
[0188] According to one specific embodiment, the plant cell is an embryogenic cell.
[0189] According to one specific embodiment, the plant cell is a monocellular embryogenetic cell.
[0190] As used herein, the term "plant gene" refers to any gene in the plant, such as an endogenous gene, which may be modified to impart silencing specificity against a harmful organism gene.
[0191] According to one embodiment, the plant gene is a non-coding gene (e.g., a non-protein-coding gene).
[0192] According to one embodiment, the plant gene is a coding gene (e.g., a protein-coding gene).
[0193] According to one embodiment, the plant gene (i.e., the predetermined sequence exhibiting homology with the nucleic acid sequence of the harmful organism gene) does not encode a silencing molecule.
[0194] According to one embodiment, the plant gene does not encode a molecule (e.g., an RNA molecule, such as a non-coding RNA molecule, as discussed in detail below) that has an intrinsic silencing activity.
[0195] According to one embodiment, the plant gene encodes a molecule (e.g., an RNA molecule, such as a non-coding RNA molecule, as discussed in detail below) that has an intrinsic silencing activity.
[0196] As used herein, the term "pest" refers to an organism that directly or indirectly harms the plant. A direct effect includes, for example, feeding on the plant's leaves. An indirect effect includes, for example, the transmission of a disease agent (e.g., a virus, a bacterium, etc.) to the plant. In the latter case, the pest is a vector for the transmission of a pathogen.
[0197] According to some embodiments, the pest is an invertebrate pest, including invertebrate pests sensitive to long dsRNAs by methods such as, but not limited to, ingestion and / or soaking. Each possibility represents a separate embodiment of the invention. According to some embodiments, an invertebrate pest is sensitive to long dsRNAs of 26 bp or more (possibly 26 to 50 bp). Each possibility represents a separate embodiment of the invention.
[0198] According to one embodiment, the invasive organism is an invertebrate organism.
[0199] Several exemplary pests include, but are not limited to, insects, nematodes, snails, slugs, spiders, caterpillars, scorpions, mites, ticks, and fungi.
[0200] Several harmful organisms include, but are not limited to, those from the orders Coleoptera (e.g., beetles), Diptera (e.g., flies, mosquitoes), Hymenoptera (e.g., sawflies, wasps, bees, and ants), Lepidoptera (e.g., butterflies and moths), Mallophaga (e.g., lice, such as chewing lice, biting lice, and bird lice), Hemiptera (e.g., true bedbugs), Homoptera including the suborder Thoracrosae (e.g., aphids, whiteflies, and scale insects), and Auchenorrhyncha (e.g., cicadas). Insects including leafhoppers, treehoppers, planthoppers, and spittlebugs; Coleorrhyncha (e.g., bryozoans and beetles); Orthoptera (e.g., grasshoppers, locusts, and crickets, including grasshoppers and crickets); Thysanoptera (e.g., thrips); Dermaptera (e.g., ear borers); Isoptera (e.g., termites); Anoplura (e.g., louses); Siphonoptera (e.g., fleas); and Trichoptera (e.g., caddisflies).
[0201] The pests of this invention include, but are not limited to, corn: European corn borer (Ostrinia nubilalis); cabbage looper (Agrotis ipsilon); corn ear borer (Helicoverpa zea); fall armyworm (Spodopterafrugiperda); southwestern corn borer (Diatraea grandiosella); small corn stalk borer (Elasmopalpuslignosellus); sugarcane borer (Diatraea saccharalis); western corn root borer (Diabrotica virgifera); northern corn root borer (Diabrotica longicornis barberi); southern corn root borer (Diabrotica undecimpunctata howardi); wireworms (Melanotus spp.); northern masked beetle (Cyclocephala borealis); southern masked beetle (Cyclocephala mmaculata); Japanese beetle (Popillia) (japonica); Corn flea beetle (Chaetocnema pulicaria); Corn salamander (Sphenophorus maidis); Corn leaf aphid (Rhopalosiphum maidis); Corn root aphid (Anuraphis maidiradicis); Salamander (Blissus leucopterus leucopterus); Red-legged grasshopper (Melanoplus femurrubrum); Migratory grasshopper (Melanoplus sanguinipes); Corn maggot (Hylemyaplatura); Corn leaf miner (Agromyza parvicornis); Thrips (Anaphothrips obscrurus); Stealing ant (Solenopsis milesta); Two-spotted spider mite (Tetranychus urticae); Sorghum: Sorghum borer (Chilopartellus); Fall armyworm (Spodoptera frugiperda); Corn ear borer (Helicoverpa zea); small corn stalk borer (Elasmopalpus lignosellus); granular worm (Feltia subterranea); white grub (Phyllophagacrinita); nematodes (Eleodes, Conoderus, and Aeolus spp.); grain leaf beetle (Oulema melanopus);Corn flea beetle (Chaetocnema pulicaria); corn salamander (Sphenophorus maidis); corn leaf aphid (Rhopalosiphum maidis); yellow sugarcane aphid (Sipha flava); salamander (Blissus leucopterus leucopterus); sorghum midge (Contarinia sorghicola); red spider mite (Tetranychus cinnabarinus); two-spotted spider mite (Tetranychus urticae); wheat armyworm (Pseudaletia unipunctata); fall armyworm (Spodoptera frugiperda); small corn stalk borer (Elasmopalpus lignosellus); western cutworm (Agrotis orthogonia); Elasmopalpus lignosellus (Elasmopalpus lignosellus); grain leaf beetle (Oulema) (melanopus); Clover weevil (Hyperapunctata); Southern corn root borer (Diabrotica undecimpunctata howardi); Russian wheat aphid; Green scale insect (Schizaphis graminum); British grain aphid (Macrosiphum avenae); Red-legged grasshopper (Melanoplus femurrubrum); Differential grasshopper (Melanoplus Differentis); Migratory grasshopper (Melanoplus sanguinipes); Black forest gall midge (Mayetiola destructor); Wheat midge (Sitodiplosis mosellana); Wheat stem maggot (Meromyza americana); Wheat bulb fly (Hylemya coarctate); Tobacco thrips (Frankliniella fusca); Wheat stem sawfly (Cephus cinctus); Wheat leafroller mite (Aceria tulipae); Sunflower: Sunflower bud moth (Suleima) helianthana); sunflower moth (Homoeosomalectellum); sunflower beetle (zygogramma exclamationis); carrot beetle (Bothyrus gibbosus); sunflower seed midge (Neolasioptera murtfeldtiana); cotton: cotton bollworm (Heliothis virescens); cotton bollworm (Helicoverpa zea);Beet armyworm (Spodoptera exigua); Pink bollworm (Pectinophoragossypiella); Cotton boll weevil (Anthonomus grandis); Cotton aphid (Aphis gossypii); Cotton flea (Pseudatomoscelis seriatus); Winged whitefly (Trialeurodes abutilonea); Bedbug (Lygus lineolaris); Red-legged grasshopper (Melanoplus femurrubrum); Differential grasshopper (Melanoplus Differentis); Onion thrips (Thrips tabaci); Tobacco thrips (Franklinkiella fusca); Carmine spider mite (Tetranychus cinnabarinus); Two-spotted spider mite (Tetranychus urticae); Rice: Sugarcane borer (Diatraea saccharalis); Fall armyworm (Spodoptera frugiperda; corn ear borer (Helicoverpazea); grape leaf beetle (Colaspis brunnea); rice water weevil (Lissorhoptrus oryzophilus); rice weevil (Sitophilus oryzae); rice leafhopper (Nephotettix nigropictus); salamander (Blissus leucopterus leucopterus); green bedbug (Acrosternum hilare); soybean: soybean leafhopper (Pseudoplusia includens); velvet caterpillar (Anticarsia gemmatalis); green clover (Plathypena scabs); European corn borer (Ostrinia nubilalis); black cutworm (Agrotis ipsilon); beet armyworm (Spodoptera exigua); cotton weevil (Heliothis virescens); cotton bollworm (Helicoverpazea); Mexican bean beetle (Epilachna) varivestis; green peach aphid (Myzus persicae); potato leafhopper (Empoasca fabae); green bedbug (Acrosternum hilare); red-legged grasshopper (Melanoplus femurrubrum); differential grasshopper (Melanoplus Differentis); corn maggot (Hylemya platura); soybean thrips (Sericothrips variabilis);Onion thrips (Thrips tabaci); Strawberry spider mite (Tetranychus turkestani); Two-spotted spider mite (Tetranychus urticae); Barley: European corn borer (Ostrinianubilalis); Black cutworm (Agrotis ipsilon); Green scale insect (Schizaphis graminum); Salamander (Blissus leucopterus leucopterus); Green bedbug (Acrosternum hilare); Brown bedbug (Euschistus servus); Corn maggot (Delia platura); Black forest gall midge (Mayetiola destructor); Brown wheat mite (Petrobia Latens); Rapeseed: Cabbage aphid (Brevicoryne brassicae); Fleas beetle (Phyllotretacruciferae); Broad-necked armyworm (Mamestra configurata); Diamondback moth (Plutella) xylostella); root maggot (Delia ssp.). ;
[0202] Exemplary nematodes include, but are not limited to, burrowing nematodes (Radopholus similis), Caenorhabditis elegans, Arabica coffee burrowing nematode (Radopholus arabocoffeae), coffee root rot nematode (Pratylenchus coffeae), root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp. and Globodera spp.), root lesion nematodes (Pratylenchus spp.), sweet potato stem nematodes (Ditylenchus dipsaci), and pine wilt nematodes. The nematode includes pine wood nematode (Bursaphelenchus xylophilus), reniform nematode (Rotylenchulus reniformis), dagger nematode (Xiphinemaindex), pseudo-root nodule nematode (Nacobbusaberrans), and leaf bud nematode (Aphelenchoides besseyi).
[0203] Exemplary fungi include, but are not limited to, *Fusarium oxysporum*, *Leptosphaeria maculans* (and *Phoma lingam*), *Sclerotinia sclerotiorum*, *Pyricularia grisea*, *Gibberella fujikuroi*, *Fusarium moniliforme*, *Magnaportheoryzae*, *Botrytis cinereal*, *Puccinia spp.*, *Fusarium graminearum*, *Blumeria graminis*, *Mycosphaerella graminicola*, *Colletotrichum spp.*, and *Ustilago*. Maydis, Melampsoralini, Phakopsorapachyrhizi, and Rhizoctonia solani.
[0204] According to a specific embodiment, the pests are ants, termites, bees, wasps, caterpillars, crickets, locusts, beetles, snails, slugs, nematodes, bedbugs, flies, fruit flies, whiteflies, mosquitoes, grasshoppers, planthoppers, borers, aphids, scale insects, thrips, spiders, mites, psyllids, ticks, moths, worms, and scorpions at different stages of their life cycle.
[0205] According to one specific embodiment, the pest is at any stage of its life cycle.
[0206] According to one embodiment, the harmful organism is a virus.
[0207] The phrase “silencing a pest gene” means reducing the expression level of a polynucleotide or the polypeptide encoded by it by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to a pest gene that is not targeted by a long dsRNA molecule designed in this invention.
[0208] Several assays for measuring the expression level of a polynucleotide or the polypeptide it encodes include, but are not limited to, RT-PCR, Western blot, immunohistochemistry and / or flow cytometry, sequencing, or any other detection method (discussed further below).
[0209] Preferably, silencing the pest's gene causes inhibition, control, and / or death of the pest, thereby limiting the damage the pest causes to the plant. Controlling a pest includes, but is not limited to, killing the pest, inhibiting its development, altering its reproductive capacity or growth to reduce the damage the pest causes to the plant, decrease the number of offspring produced, create unsuitable pests, create pests more vulnerable to predators, or prevent the pest from consuming the plant.
[0210] As used herein, the term "pest gene" refers to any gene in the pest that is essential for its growth, development, reproduction, or infectivity. The gene may be expressed in any tissue of the pest; however, in one specific embodiment, several genes that target and inhibit the pest are expressed in several cells of the pest's intestinal tissue, several cells of the pest's midgut, several cells of the intestinal lumen or midgut lining, several cells of the pest's gut microbiome, and several cells of the pest's immune system. Such target genes may be involved in, for example, intestinal cell metabolism, growth, differentiation, and the immune system.
[0211] The exemplary harmful organism genes targeted by this method include, but are not limited to, the genes listed in Tables 1A to 1B below.
[0212] According to one specific embodiment, the nematode genes include several genes similar to those in Radopholus similis, such as calreticulin 13 (CRT) or collagen 5 (col-5).
[0213] According to a specific embodiment, the fungal genes include several genes from Fusarium oxysporum, including FOW2, FRP1, and OPR.
[0214] According to one embodiment, compared to a plant damaged by the pest and not treated with the long dsRNA molecule designed in this invention, silencing a pest gene reduces several disease symptoms in a plant or reduces damage to the plant (caused by the pest) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.
[0215] Several assays for controlling a pest are well known in the art, see, for example, U.S. Patent No. 5,614,395, which is incorporated herein by reference. These techniques include measuring the average lesion diameter, pathogen biomass, and the overall percentage of decayed plant tissues over time. For example, see Thomma et al. (1998) Plant Biology 95:15107-15111, which is incorporated herein by reference. See also Baum et al. (2007) Nature Biotech 11:1322-1326 and WO 2007 / 035650, which provide several whole-plant feeding trials and several maize root feeding trials.
[0216] According to one embodiment, the method includes selecting a nucleic acid sequence of a plant gene that exhibits a predetermined sequence homology with a nucleic acid sequence of a pest gene.
[0217] According to one embodiment, the sequence homology between the nucleic acid sequence of the plant gene and the nucleic acid sequence of the harmful organism gene includes 60% to 100%, 70% to 80%, 70% to 90%, 70% to 100%, 75% to 100%, 80% to 90%, 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identity.
[0218] According to a specific embodiment, the sequence homology includes 75% to 100% identity between the nucleic acid sequence of the plant gene and the nucleic acid sequence of the pest gene.
[0219] According to a specific embodiment, the sequence homology includes 85% to 100% identity between the nucleic acid sequence of the plant gene and the nucleic acid sequence of the pest gene.
[0220] According to a specific embodiment, the sequence homology includes 75% to 100% identity between the nucleic acid sequence of the plant gene and the nucleic acid sequence of the pest gene.
[0221] According to one embodiment, the sequence homology includes at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity between the nucleic acid sequence of the plant gene and the nucleic acid sequence of the pest gene.
[0222] Homology (e.g., percentage of homology, sequence identity + sequence similarity) can be determined using any homology comparison software that calculates it as a pairwise sequence alignment.
[0223] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes the number of residues in two sequences that are identical when aligned. When the sequence identity percentage is applied to several proteins, it should be recognized that the positions of several dissimilar residues are often due to several conserved amino acid substitutions, in which several amino acid residues are replaced by several other amino acid residues having several similar chemical properties (e.g., charge or hydrophobicity) without altering the functional properties of the molecule. When several sequences differ in terms of several conserved substitutions, the sequence identity percentage can be adjusted upwards to correct for the conservatism of the substitutions. Several sequences that differ through such conserved substitutions are considered to have “sequence similarity” or “similarity.” Several means of making such adjustments are well known to those skilled in the art. Generally, this involves rating a conserved substitution as a partial mismatch rather than a full mismatch, thereby increasing the sequence identity percentage. Therefore, for example, if the score for an identical amino acid is 1 and the score for a non-conservative substitution is 0, the score for a conserved substitution is between 0 and 1. The scores for several conserved substitutions are calculated, for example, according to the algorithm of Henikoff S and Henikoff JG (Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci., USA, 1992, 89(22): 10915-9).
[0224] Any homology comparison software can be used, including, for example, BlastN software from the National Center for Biotechnology Information (NCBI), to determine identity (e.g., percentage of homology) by using several default parameters.
[0225] According to some embodiments of the present invention, the identity is a global identity, that is, the identity of the entire amino acid or several nucleic acid sequences of the present invention, rather than the identity of several parts thereof.
[0226] According to some embodiments of the present invention, the terms "homology" or "homologous" refer to the identity of two or more nucleic acid sequences; or the identity of two or more amino acid sequences; or the identity of one amino acid sequence with one or more nucleic acid sequences.
[0227] According to some embodiments of the present invention, the homology is a global homology, that is, the homology of the entire amino acid or nucleic acid sequence of the present invention, rather than the homology of several parts thereof.
[0228] The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools. The following is a non-limiting description of such tools that can be used with some embodiments of the present invention.
[0229] When starting with a polynucleotide sequence and comparing it with other polynucleotide sequences, the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available from emboss(dot)sourceforge(dot)net / apps / cvs / emboss / apps / needle(dot)html) can be used with the following default parameters: (EMBOSS-6.0.1) gapopen=10; gapextend=0.5; data file=EDNAFULL; brief=YES.
[0230] According to some embodiments of the present invention, the parameters used with the EMBOSS-6.0.1 Needleman-Wunsch algorithm are: gapopen = 10; gapextend = 0.2; data file = EDNAFULL; brief = YES.
[0231] According to some embodiments of the present invention, the threshold for determining homology using the EMBOSS-6.0.1 Needleman-Wunsch algorithm to compare several polynucleotides is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[0232] According to some embodiments, the determination of the degree of homology also requires the use of the Smith-Waterman algorithm (for protein-protein or nucleotide-nucleotide comparisons).
[0233] The default parameters for the GenCore 6.0 Smith-Waterman algorithm include: model = sw.model.
[0234] According to some embodiments of the present invention, the threshold for determining homology using the Smith-Waterman algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[0235] According to some embodiments of the invention, before performing global homology with the polypeptide or polynucleotide of interest (e.g., 80% global homology across the entire sequence), the overall homology is performed on sequences pre-selected by local homology with the polypeptide or polynucleotide of interest (e.g., 60% identity over more than 60% of the sequence length). For example, several homologous sequences are selected using BLAST software, where the Blastp and tBlastn algorithms serve as several filters in the first stage, and the needle (EMBOSS package) or Frame+ algorithm alignment is used in the second stage. The definition of local identity (several BLAST alignments) is very loose—60% identity over 60% of the sequence length span—because it is only used as one filter in the global alignment stage. In the specific embodiment (when using local identity), the default filter of the Blast package (by setting the parameter "-FF") is not used.
[0236] In the second stage, several homologs are defined based on at least 80% global identity with the core gene polypeptide sequence. According to some embodiments, this homology is a local homology or a local identity.
[0237] Several local alignment tools include, but are not limited to, the BlastP, BlastN, BlastX, or TBLASTN software from the National Center for Biotechnology Information (NCBI), the FASTA algorithm, and the Smith-Waterman algorithm.
[0238] According to a specific embodiment, homology is determined using BlastN with the following parameters: maximum target sequences = 1000, expected threshold = 10, word size = 11, matching score = 2, mismatch score = -3, gap existence cost = 5, and gap expansion cost = 2.
[0239] According to a specific embodiment, a nucleic acid sequence of a plant gene exhibiting homology to a predetermined sequence of a nucleic acid sequence of the harmful organism gene is selected by identifying several plant transcripts that have "several homology stretches" with the harmful organism transcript. According to a specific implementation, the homology stretches are 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, ... 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10,000 or more nucleotides (e.g., 20 to 50 nucleotides, 20 to 25 nucleotides, e.g., 21 nucleotides). Within the 20 to 50 nucleotides (e.g., 21 nucleotides), the homology between the plant transcript and the harmful organism transcript is preferably 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
[0240] According to a specific embodiment, when the harmful organism is a Heterodera glines, the harmful organism's gene is as described in accession number AF469060.1 (Heterodera glycinesubiquitin extension protein), and the plant gene is as described in NM_001203752.2 (Arabidopsis thaliana ubiquitin 11 (UBQ11)).
[0241] According to a specific embodiment, when the harmful organism is a Heterodera glines, the harmful organism's genes are as described in accession number AF500024.1 (Heterodera glycinesputative gland protein G8H07) and the plant genes are as described in NM_116351.7 (Arabidopsis thaliana glycosyl transferase family 1 protein (AT4G01210)).
[0242] According to a specific embodiment, when the harmful organism is a nematode (Heteroderaglycines), the harmful organism's genes are as described in accession number AF502391.1 (Heteroderaglycines putative gland protein G10A06), and the plant genes are as described in NM_001037071.1 (Arabidopsis thaliana bZIP transcription factor family protein, TGA1).
[0243] According to one embodiment, the method includes modifying a plant endogenous nucleic acid sequence encoding an RNA molecule to impart silencing specificity to the plant gene, such that several small RNA molecules capable of recruiting RNA-dependent RNA polymerase (RdRp) form base complementarity with a transcript of the plant gene, the several small RNA molecules being derived from RNA addition, to produce the long dsRNA molecule capable of silencing the harmful organism gene.
[0244] According to one embodiment, the RNA molecule is a non-coding RNA molecule.
[0245] As used herein, the term “non-coding RNA molecule” refers to an RNA sequence that has not been translated into an amino acid sequence and does not encode a protein.
[0246] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a non-coding gene (e.g., a non-protein-coding gene). Exemplary non-coding portions of the genome include, but are not limited to, several introns, several genes of several non-coding RNAs, several DNA methylation regions, several enhancers and several locus control regions, several insulators, several S / MAR sequences, several non-protein-coding pseudogenes, several transposons, several non-autonomous transposable elements (e.g., Alu, SINES, and several mutated non-coding transposons and retrotransposons), and several simple repeats in several centromere and telomere regions of chromosomes.
[0247] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a universally expressed non-coding gene.
[0248] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a non-coding gene expressed in a tissue-specific manner (e.g., in a leaf, fruit, or flower).
[0249] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a non-coding gene expressed in an inducible manner.
[0250] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a non-coding gene that is developmentally regulated.
[0251] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located between several genes, i.e., in the intergenic region.
[0252] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within an intron of a non-coding gene.
[0253] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a coding gene (e.g., a protein-coding gene).
[0254] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in an exon of a coding gene (e.g., a protein-coding gene).
[0255] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in an exon of a nontranslated region (UTR) encoding a coding gene (e.g., a protein-coding gene).
[0256] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within a translational exon of a coding gene (e.g., a protein-coding gene).
[0257] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within an intron of a coding gene (e.g., a protein-coding gene).
[0258] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within a universally expressed coding gene.
[0259] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within a coding gene expressed in a tissue-specific manner (e.g., in a leaf, fruit, or flower).
[0260] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located within a coding gene expressed in an inducible manner.
[0261] According to one embodiment, the nucleic acid sequence encoding the RNA molecule is located in a gene encoding developmental regulation.
[0262] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is typically affected by the RNA silencing processing mechanism or activity. However, this document also considers some changes of several nucleotides (e.g., for miRNAs of up to 24 nucleotides) that could trigger a processing mechanism leading to RdRP recruitment, RNA interference, or translational repression.
[0263] According to one specific embodiment, the RNA molecule is endogenous to the plant cell (naturally occurring, for example, naturally occurring). It should be understood that the RNA molecule may also be exogenous to the cell (i.e., externally added and not naturally occurring in the plant cell).
[0264] According to some embodiments, the RNA molecule (e.g., a non-coding RNA molecule) includes an intrinsic translational repression activity.
[0265] According to some embodiments, RNA molecules (e.g., non-coding RNA molecules) include an intrinsic RNA interference (RNAi) activity.
[0266] According to some embodiments, RNA molecules (e.g., non-coding RNA molecules) do not include an intrinsic translational repression activity or an intrinsic RNAi activity (i.e., non-coding RNA molecules do not have an RNA silencing activity).
[0267] According to one embodiment of the invention, the RNA molecule (e.g., a non-coding RNA molecule) is specific to a natural plant RNA (e.g., natural plant RNA) and does not cross-inhibit or silence a harmful biological RNA or the plant RNA of interest (i.e., a transcript of the plant gene), unless designed to do so (as described below) show 100% or less global homology with the target gene, for example, less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81% global homology with the target gene; as determined at the RNA or protein level by RT-PCR, Western ink dot assay, immunohistochemistry and / or flow cytometry, sequencing, or any other detection method.
[0268] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is an RNA silencing or RNA interference (RNAi) molecule (also known as a "silencing molecule").
[0269] The term “RNA silencing” or RNAi refers to a cellular regulatory mechanism in which several non-coding RNA molecules (“RNA silencing molecules,” “silencing molecules,” or “RNAi molecules”) mediate gene expression or translation in a sequence-specific manner, either through co-transcriptional or post-transcriptional repression.
[0270] As used herein, a "silencing molecule capable of recruiting RNA-dependent RNA polymerase (RdRp)" means a silencing molecule capable of binding RdRp to the site where it interacts with the target transcript, thereby enabling the formation of a long dsRNA based on another RNA molecule as a template. In a non-limiting example, the silencing molecule capable of recruiting RdRp is a miRNA, such as, but not limited to, a 22 nt long miRNA, and a TAS transcript is used as a template for the miRNA / RISC / RdRp complex, thereby producing a long dsRNA based on the TAS transcript.
[0271] According to one embodiment, the RNA molecule (e.g., an RNA silencing molecule) is capable of mediating RNA repression during transcription (co-transcriptional gene silencing).
[0272] According to one specific embodiment, co-transcriptional gene silencing includes epigenetic silencing (e.g., a chromatic state that prevents the expression of functional genes).
[0273] According to one embodiment, the RNA molecule (e.g., an RNA silencing molecule) is capable of mediating RNA repression after transcription (post-transcriptional gene silencing).
[0274] Post-transcriptional gene silencing (PTGS) is generally the process of degradation or cleavage of a number of messenger RNA (mRNA) molecules (usually occurring in the cytoplasm), which reduces their activity by preventing translation. For example, and as discussed in detail below, a leader strand of an RNA silencing molecule pairs with a complementary sequence in an mRNA molecule and induces cleavage via, for example, Argonaute 2 (Ago2).
[0275] Co-transcriptional gene silencing generally refers to the inactivation of gene activity (i.e., transcriptional repression), and typically occurs in the cell nucleus. This gene activity repression is mediated by several epigenetic-related factors, such as several methyltransferases, methylate target DNA, and histones. Therefore, in co-transcriptional gene silencing, the association between a small RNA and a target RNA (small RNA transcriptional interaction) disrupts the stability of the target nascent transcript and recruits several DNA and histone modifying enzymes (i.e., several epigenetic factors) that induce chromatin remodeling into a structure that represses gene activity and transcription. Furthermore, in co-transcriptional gene silencing, several chromatin-associated long non-coding RNA scaffolds can independently recruit several chromatin-modifying complexes, separate from several small RNAs. These co-transcriptional silencing mechanisms form several RNA surveillance systems that detect and silence several inappropriate transcriptional events and provide a storage of these events through several self-reinforcing epigenetic loops (as described in the following literature: D. Hoch and D. Moazed, RNA-mediated epigenetic regulation of gene expression, Nat Rev Genet. (2015) 16(2): 71–84).
[0276] According to one embodiment of the present invention, the RNAi biogenesis / processing machinery produces the RNA silencing molecule.
[0277] According to one embodiment of the present invention, the RNAi biogenesis / processing machinery produces the RNA silencing molecule, but a specific target has not yet been identified.
[0278] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is capable of inducing RNA interference (RNAi).
[0279] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or an RNA silencing molecule) is processed from a precursor.
[0280] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from a single-stranded RNA (ssRNA) precursor.
[0281] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from a single-stranded RNA precursor with a double-stranded structure.
[0282] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from a dsRNA precursor (e.g., comprising perfect and imperfect base pairings).
[0283] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from an unstructured RNA precursor.
[0284] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from a protein-coding RNA precursor.
[0285] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed from an RNA precursor.
[0286] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or the RNA silencing molecule) is processed and bound to an RNA-induced silencing complex (RISC).
[0287] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule or an RNA silencing molecule) is processed and bound to an RNAi processing mechanism, such as a ribonuclease, including but not limited to Dicer, Ago2, the DICER protein family (e.g., DCR1 and DCR2), the Dicer-like protein family (e.g., DCL1, DCL2, DCL3, DCL4), the ARGONAUTE protein family (e.g., AGO1, AGO2, AGO3, AGO4), several tRNA cleaving enzymes (e.g., RNY1, angiopoietin, RNase P, RNase P-like, SLFN3, ELAC1, and ELAC2), and Piwi-interacting RNA (piRNA)-related proteins (e.g., AGO3, AUBERGINE, HIWI, HIWI2, HIWI3, PIWI, ALG1, and ALG2) (as further discussed below).
[0288] According to one embodiment, dsRNA may originate from two different complementary RNAs, or from a single RNA that folds itself to form dsRNA.
[0289] The following is a detailed description of several RNA silencing molecules (e.g., non-coding RNA molecules) that bind to an RNA-induced silencing complex (RISC) and include an intrinsic RNAi activity (e.g., several RNA silencing molecules) that can be used according to several specific embodiments of the invention.
[0290] Based on the activity of fully and incompletely paired RNAs (i.e., double-stranded RNA; dsRNA), siRNA, and shRNA—the presence of several long dsRNAs in several cells stimulates the activity of a ribonuclease III enzyme (called dicer). Dicer (also known as the ribonuclease Dicer or a helicase with an RNase motif) is an enzyme commonly referred to as a Dicer-like (DCL) protein in several plants. Different plants have different numbers of DCL genes; therefore, for example, the Arabidopsis genome typically has four DCL genes, rice has eight, and the maize genome has five. Dicer is involved in the process of processing the dsRNA into several short fragments of dsRNA (called short interfering RNA (siRNA)). The length of siRNA derived from dicer activity is typically about 21 to about 23 nucleotides and includes a double strand of about 19 base pairs with two 3' nucleotide overhangs.
[0291] According to one embodiment, several dsRNA precursors longer than 21 bp are used. Various studies have shown that long dsRNAs can be used to silence gene expression without inducing stress responses or causing significant off-target effects—see, for example, Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13: 3803-3810; Bhargava A et al., BrainRes.Protoc., 2004, 13: 115-125; Diallo M. et al., Oligonucleotides, 2003, 13: 381-392; Paddison PJ et al., Proc.Natl Acad.Sci.USA, 2002; 99: 1443-1448; Tran N. et al., FEBS Lett., 2004, 573: 127-134.
[0292] The term "siRNA" refers to a small, repressive RNA duplex (typically between 18 and 30 base pairs) that induces the RNA interference (RNAi) pathway. Typically, siRNA is chemically synthesized as 21-mers with a central 19 bp duplex region and symmetrical 2-base 3'-overhangs at the ends, although it has recently been described that chemically synthesized 25- to 30-base-long RNA duplexes can be up to 100-fold more potent than 21-mers at the same position. This suggests that the observed increased potency obtained by using longer RNA to trigger RNAi is due to providing a matrix (27-mers) to Dicer rather than a product (21-mers), which increases the speed or efficiency of the siRNA duplex entering the RISC.
[0293] It has been found that the position (rather than the composition) of the 3'-protrusion affects the potency of an siRNA, and that asymmetric duplexes with a 3'-protrusion on the antisense strand are generally more potent than those with the 3'-protrusion on the sense strand (Rose et al., 2005).
[0294] Several strands of a double-stranded interfering RNA (e.g., siRNA) can be linked to form a hairpin or stem-loop structure (e.g., shRNA). Therefore, as described above, the RNA silencing molecule in some embodiments of the present invention can also be a short hairpin RNA (shRNA).
[0295] As used herein, the term short hairpin RNA (“shRNA”) refers to an RNA molecule having a stem-loop structure, comprising a first and a second region of complementary sequences, the complementarity and orientation of said regions being sufficient to allow base pairing to occur between said regions, said first and second regions being connected by a loop region, said loop being due to the lack of base pairing between said loop nucleotides (or said nucleotide analogs) within said loop region. The number of said loop nucleotides is between and includes 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11, and some of said loop nucleotides may participate in base pairing interactions with other nucleotides in said loop. Examples of said loop oligonucleotide sequences that may be used to form said loop include 5'-CAAGAGA-3 and 5'-UUACAA-3' (International Patent Applications WO2013126963 and WO2014107763). Those skilled in the art will recognize that the resulting single-stranded oligonucleotides form a stem-loop or hairpin structure, including a double-stranded region capable of interacting with the RNAi mechanism.
[0296] The RNA silencing molecules described in some embodiments of the present invention are not limited to those containing only RNA, but further include several chemically modified nucleotides and non-nucleotides.
[0297] This invention considers various types of siRNA, including trans-acting siRNA (TasiRNA), repeat-associated siRNA (Ra-siRNA), and natural-antisense transcript-derived siRNA (Nat-siRNA).
[0298] According to one specific embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a phased small interfering RNA (phasiRNA). Several “phasiRNAs” originate from an mRNA that is converted from RDR6 to dsRNA and processed by DCL4, such as several trans-acting siRNAs (several tasiRNAs) in Arabidopsis (Vazquez et al., 2004). In a particular case, several phasiRNAs may also be the 24-nucleotide product of DCL5 (formerly known as DCL3b) in several herbaceous reproductive tissues (Song et al., 2012). The trans-acting names (tasiRNAs) of some phasiRNAs derive from their ability to function like several miRNAs in a homology-dependent manner, leading to AGO1-dependent silencing of several mRNAs from several genes whose mRNAs are not their origin (see below).
[0299] According to one specific embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a tasiRNA. "TasiRNA" is a class of secondary siRNAs generated in a phased pattern from several non-coding TAS transcripts triggered by miRNA (Peragine et al., 2004; Vazquez et al., 2004; Allen et al., 2005; Yoshikawa et al., 2005). The term "phased" simply means that the several small RNAs are precisely generated from a specific nucleotide in a head-to-tail arrangement; this configuration is caused by miRNA-triggered initiation followed by DCL4-catalyzed cleavage. The key proteins involved in tasiRNA biogenesis include, but are not limited to, RDR6, the suppressor of gene silencing 3 (SGS3), DCL4, AGO1, AGO7, and double-stranded RNA binding factor 4 (Peragine et al., 2004; Vazquez et al., 2004; Xie et al., 2005; Adenot et al., 2006; Montgomery et al., 2008a; Fukudome et al., 2011). Most importantly, there are two mechanisms for the production of several 21-nucleotide tasiRNAs, referred to as the “one-hit” or “two-hit” pathways. In the one-hit mechanism, a single miRNA directs the cleavage of the mRNA target, triggering the production of several phasiRNAs in fragment 39 to reach (or downstream) the target site (Allen et al., 2005). The one-hit miRNA trigger is typically 22 nucleotides long (Chen et al., 2010; Cuperus et al., 2010). In the double-hit model, a pair of 21-nucleotide miRNA target sites is used, where cleavage occurs only at 39 target sites, triggering the generation of several phasiRNA fragments (or upstream of the target sites) (Axtell et al., 2006).
[0300] According to one embodiment, the silent RNA comprises “piRNA,” which is a class of Piwi-interacting RNAs approximately 26 to 31 nucleotides in length. Several piRNAs typically form several RNA-protein complexes by interacting with several Piwi proteins; that is, several antisense piRNAs are typically loaded into several Piwi proteins (e.g., Piwi, Ago3, and Aubergine (Aub)).
[0301] miRNA - According to another embodiment the RNA silencing molecule may be a miRNA. According to another embodiment, the RNA silencing molecule can be a miRNA.
[0302] The terms “microRNA,” “miRNA,” and “miR” are synonyms referring to a collection of several non-coding single-stranded RNA molecules, approximately 19 to 24 nucleotides in length, that regulate gene expression. Several miRNAs are widely distributed in various organisms (e.g., insects, mammals, plants, nematodes) and have been shown to play roles in development, homeostasis, and disease pathogenesis.
[0303] Initially, the pre-miRNA exists as a long, incompletely double-stranded stem-loop RNA, which is further processed by Dicer into an siRNA-like double-stranded RNA, comprising a mature guide strand (miRNA) and a similarly sized segment (called the passenger strand (miRNA*)). The miRNA and miRNA* can be derived from the opposite arms of the pri-miRNA and pre-miRNA. Several miRNA* sequences can be found in libraries of cloned miRNAs, but their occurrence rate is generally lower than that of miRNAs because they are mostly non-functional and undegraded in the cell.
[0304] Although initially present as a double-stranded form with miRNA*, the miRNA eventually becomes a single-stranded RNA incorporated into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). Various proteins can form the RISC, which can cause specificity for the miRNA / miRNA* duplex, the binding site of the target gene, the activity of the miRNA (inhibition or activation), and variability in which strand of the miRNA / miRNA* duplex is loaded into the RISC.
[0305] When the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* is removed and degraded. The miRNA:miRNA* duplex loaded into the RISC has a loosely paired 5' end. When the miRNA:miRNA* has approximately equal 5' pairing at both ends, both the miRNA and miRNA* may possess gene silencing activity.
[0306] The RISC identifies several target nucleic acids based on the high complementarity between the miRNA and the mRNA, particularly through positions 2 to 8 of the miRNA (referred to as the "seed sequence").
[0307] Numerous studies have focused on the base-pairing requirements between miRNAs and their mRNA targets for efficient translational repression (reviewed by Bartel (2004, Cell, 116–281)). Several computational studies analyzing miRNA binding across the genome have shown a specific role for bases 2–8 at the 5' end of the miRNA (also known as the “seed sequence”) in target binding, but the role of the first nucleotide, typically “A”, has also been identified (Lewis et al., 2005, Cell, 120–15). Similarly, Krek et al. used nucleotides 1–7 or 2–8 to identify and validate multiple targets (2005, Nat Genet., 37–495). These multiple target sites in the mRNA can be located in the 5' UTR, 3' UTR, or coding regions. Interestingly, multiple miRNAs can regulate the same mRNA targets by recognizing the same or multiple sites. The presence of several miRNA binding sites in most genetically recognized targets may indicate that the synergistic action of several RISCs provides the most effective translational repression.
[0308] miRNAs may guide the regulation of gene expression under RISC through one of two mechanisms: mRNA cleavage or translation repression. If the mRNA and the miRNA have a certain degree of complementarity, the miRNA can direct the cleavage of the mRNA. When a miRNA directs cleavage, the cut typically occurs between several nucleotides that pair with residues 10 and 11 of the miRNA. Alternatively, if the miRNA and the miRNA do not have the necessary complementarity, the miRNA can repress translation. Translation repression is likely more prevalent in animals due to the lower degree of complementarity between the miRNA and its binding site.
[0309] It should be noted that variability may exist at the 5' and 3' ends of any pair of miRNAs and miRNA*. This variability may be due to variability relative to the cleavage site during enzymatic processing by Drosha and Dicer. The variability at the 5' and 3' ends of miRNAs and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. These mismatches in the stem strands can cause a large number of different hairpin structures. The variability in the stem structures may also cause variability in the products cleaved by Drosha and Dicer.
[0310] According to one embodiment, miRNA can be processed independently of Dicer, for example, via Argonaute 2.
[0311] It should be understood that the pre-miRNA sequence may include 45 to 90, 60 to 80, or 60 to 70 nucleotides, while the pri-miRNA sequence may include 45 to 30,000, 50 to 25,000, 100 to 20,000, 1,000 to 1,500, or 80 to 100 nucleotides.
[0312] Antisense-antisense is a single-stranded RNA designed to prevent or suppress gene expression by specifically hybridizing with its mRNA. Downregulation of a target RNA can be achieved using an antisense polynucleotide that specifically hybridizes with an mRNA transcript encoding the target RNA.
[0313] Transposable RNA Several transposable genetic elements (TEs) comprise a large number of DNA sequences, all of which can move directly to several new sites in several genomes via a cut-and-paste mechanism (several transposons) or indirectly via an RNA intermediate (retrotransposon). Based on whether the TEs possess several ORFs encoding the proteins required for transposition, they are classified into several autonomous and non-autonomous categories. RNA-mediated gene silencing is one of the mechanisms by which the genome controls the activity of several TEs and exerts several detrimental effects stemming from genomic and epigenetic instability.
[0314] As mentioned above, the RNA molecule (e.g., a non-coding RNA molecule) may not include a typical (intrinsic) RNAi activity (e.g., it is not a typical canonical RNA silencing molecule, or its target has not yet been identified). Such non-coding RNA molecules include: According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a transfer RNA (tRNA). The term "tRNA" refers to an RNA molecule that is a physical link between a nucleotide sequence of several nucleic acids and an amino acid sequence of several proteins, formerly known as soluble RNA or sRNA. tRNA is typically about 76 to 90 nucleotides in length.
[0315] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a ribosomal RNA (rRNA). The term "rRNA" refers to the RNA component of the ribosome, namely a small ribosomal subunit or a large ribosomal subunit.
[0316] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a small nuclear RNA (snRNA or U-RNA). The terms "sRNA" or "U-RNA" refer to several small RNA molecules found in several splicing specks and several Cajal bodies in the nuclei of several eukaryotic cells. snRNA is typically about 150 nucleotides in length.
[0317] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a small nucleolar RNA (snoRNA). The term "snoRNA" refers to a class of small RNA molecules that primarily guide the chemical modification of other RNAs (e.g., rRNA, tRNA, and snRNA). snoRNAs are generally classified into two categories: C / D-box snoRNAs are typically about 70 to 120 nucleotides in length and are associated with methylation; while H / ACA-box snoRNAs are typically about 100 to 200 nucleotides in length and are associated with pseudouridinelation.
[0318] Similar to snoRNA are scaRNA (i.e., several small Cajal body RNA genes), which play a similar role in RNA maturation. However, their target is spliceosomal snRNA, and they perform site-specific modifications on several spliceosomal snRNA precursors (in the several Cajal bodies in the cell nucleus).
[0319] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is an extracellular RNA (exRNA). The term "exRNA" refers to several extracellular RNA species that are present in the cells through which they are transcribed (e.g., exosomal RNA).
[0320] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a repeat-derived RNA. The term "repeat-derived RNA" refers to an RNA encoded by DNA derived from several reverse genomic repeats (e.g., but not limited to DNA generated through DNA recombination, genomic site replication, several transposition events, etc.).
[0321] According to one embodiment, the RNA molecule (e.g., a non-coding RNA molecule) is a long non-coding RNA (lncRNA). The term "lncRNA" or "long ncRNA" refers to a non-protein-coding transcript that is typically longer than 200 nucleotides.
[0322] According to a specific embodiment, non-limiting examples of several RNA molecules (e.g., non-coding RNA molecules) that bind to RISC include, but are not limited to, microRNA (miRNA), piwi-interacting RNA (piRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), phased small interfering RNA (phasiRNA), trans-acting siRNA (tasiRNA), small nuclear RNA (snRNA or URNA), transposon element RNA (such as autonomous and non-autonomous transposon RNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small Cahalson RNA (scaRNA), ribosomal RNA (rRNA), extracellular RNA (exRNA), repeat-derived RNA, and long non-coding RNA (lncRNA).
[0323] According to a specific embodiment, non-limiting examples of several RNAi molecules that bind to RISC include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA).
[0324] According to one embodiment, several small RNA molecules processed from the RNA molecules (e.g., non-coding RNA molecules) of some embodiments of the present invention are capable of recruiting RNA-dependent RNA polymerase (RdRp).
[0325] The term "processed" refers to the biogenesis of RNA molecules being cleaved into smaller RNA forms that can bind to the RNA-induced silencing complex (RISC). For example, pre-miRNA is processed into a mature miRNA, for instance, via Dicer.
[0326] As used herein, the terms “small RNA form” or “several small RNAs” or “several small RNA molecules” refer to the mature small RNA, such as a transcript of the plant gene (or a fragment thereof), that is capable of hybridizing with a target RNA.
[0327] According to one embodiment, the length of the plurality of small RNAs does not exceed 250 nucleotides, for example including 20 to 250, 20 to 200, 20 to 150, 20 to 100, 20 to 50, 20 to 40, 20 to 30, 20 to 25, 20 to 26, 30 to 100, 30 to 80, 30 to 60, 30 to 50, 30 to 40, 50 to 150, 50 to 100, 50 to 80, 50 to 70, 100 to 250, 100 to 200, 100 to 150, 150 to 250, and 150 to 200 nucleotides.
[0328] According to one specific embodiment, the small RNA molecule comprises 20 to 50 nucleotides.
[0329] According to one specific embodiment, the small RNA molecule comprises 20 to 30 nucleotides.
[0330] According to one specific embodiment, the small RNA molecule comprises 21 to 29 nucleotides.
[0331] According to one specific embodiment, the small RNA molecule comprises 21 to 24 nucleotides.
[0332] According to one specific embodiment, the small RNA molecule comprises 21 nucleotides.
[0333] According to one specific embodiment, the small RNA molecule comprises 22 nucleotides.
[0334] According to one specific embodiment, the small RNA molecule comprises 23 nucleotides.
[0335] According to one specific embodiment, the small RNA molecule comprises 24 nucleotides.
[0336] According to one specific embodiment, the small RNA molecule is composed of 20 to 50 nucleotides.
[0337] According to one specific embodiment, the small RNA molecule is composed of 20 to 30 nucleotides.
[0338] According to one specific embodiment, the small RNA molecule is composed of 21 to 29 nucleotides.
[0339] According to one specific embodiment, the small RNA molecule is composed of 21 to 24 nucleotides.
[0340] According to one specific embodiment, the small RNA molecule is composed of 21 nucleotides.
[0341] According to one specific embodiment, the small RNA molecule is composed of 22 nucleotides.
[0342] According to one specific embodiment, the small RNA molecule is composed of 23 nucleotides.
[0343] According to one specific embodiment, the small RNA molecule is composed of 24 nucleotides.
[0344] According to one embodiment, the small RNA molecule includes a silencing activity (i.e., several silencing molecules).
[0345] As described above, several silencing molecules (e.g., several RNA silencing molecules) in some embodiments of the present invention are capable of recruiting RNA-dependent RNA polymerase (RdRp).
[0346] The term "RNA-dependent RNA polymerase" or "RdRp" refers to the enzyme that catalyzes the replication of RNA from an RNA template.
[0347] According to one embodiment, the small RNA molecule includes an amplifier or primer activity targeting the RdRp.
[0348] According to a specific embodiment, the silencing molecules that can recruit the RdRp are selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), trans-interacting siRNA (tasiRNA), phased small interfering RNA (phasiRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), extracellular RNA (exRNA), repetitive derived RNA, and autonomous and non-autonomous transposable RNA.
[0349] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 21 to 24 nucleotides.
[0350] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 21 nucleotides.
[0351] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 22 nucleotides.
[0352] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 23 nucleotides.
[0353] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp comprises 24 nucleotides.
[0354] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp is composed of 21 nucleotides.
[0355] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp is composed of 22 nucleotides.
[0356] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp is composed of 23 nucleotides.
[0357] According to some embodiments of the present invention, the silencing molecule capable of recruiting the RdRp is composed of 24 nucleotides.
[0358] According to a specific embodiment, the silencing molecule capable of recruiting the RdRp is miRNA.
[0359] According to one specific embodiment, the miRNA comprises a mature small RNA of 21 to 25 nucleotides. According to one specific embodiment, the miRNA comprises a mature small RNA of 21 nucleotides.
[0360] According to one specific embodiment, the miRNA comprises a mature small RNA of 22 nucleotides.
[0361] According to one specific embodiment, the miRNA comprises a mature small RNA of 23 nucleotides.
[0362] According to one specific embodiment, the miRNA comprises a mature small RNA of 24 nucleotides.
[0363] According to one specific embodiment, the miRNA comprises a mature small RNA of 25 nucleotides.
[0364] According to one specific embodiment, the miRNA is a mature small RNA of 21 to 25 nucleotides.
[0365] According to one specific embodiment, the miRNA is a mature small RNA of 21 nucleotides.
[0366] According to one specific embodiment, the miRNA comprises a mature small RNA of 22 nucleotides.
[0367] According to one specific embodiment, the miRNA is a mature small RNA of 23 nucleotides.
[0368] According to one specific embodiment, the miRNA is a mature small RNA of 24 nucleotides.
[0369] According to one specific embodiment, the miRNA is a mature small RNA of 25 nucleotides.
[0370] Exemplary miRNAs include, but are not limited to, miR-156a, miR-156c, miR-162a, miR-162b, miR-167d, miR-169b, miR-173, miR-393a, miR-393b, miR-402, miR-403, miR-447a, miR-447b, miR-447c, miR-472, miR-771, miR-777, miR-828, miR-830, miR-831, miR-833a, miR- 833a,miR-840,miR-845b,miR-848,miR-850,miR-853,miR-855,miR-856,miR-864,miR-2933a,miR-2933b,miR-2936,miR-4221, miR-5024,miR-5629,miR-5648,miR-5996,miR-8166,miR-8167a,miR-8167b,miR-8167c,miR-8167d,miR-8167e,miR-8167f,miR-81777 -8182.
[0371] As described above, the method in some embodiments of the present invention includes modifying a plant endogenous nucleic acid sequence encoding an RNA molecule to confer gene silencing specificity against the plant.
[0372] According to one embodiment, when the RNA molecule does not have an intrinsic silencing activity, the method further includes introducing a DNA editing agent that confers silencing specificity to the RNA molecule against the plant gene into the plant cell.
[0373] According to one embodiment, when the RNA molecule has an intrinsic silencing activity against a natural plant gene, the method further includes introducing a DNA editing agent into the plant cell, the DNA editing agent redirecting the silencing specificity of the RNA molecule to the plant gene, which is different from the natural plant gene.
[0374] Several methods for modifying multiple nucleic acid sequences are discussed in detail below.
[0375] According to some embodiments, such as in the second model described herein, a nucleic acid sequence of a plant gene is modified to encode a long dsRNA molecule that imparts a silencing specificity against a harmful organism gene. According to some embodiments, the nucleic acid sequence encodes an RNA molecule having an intrinsic silencing activity against a natural plant gene, such that the modification produces a silencing RNA having a new silencing activity (e.g., against a harmful organism gene) in addition to or replacing the intrinsic silencing activity. Each possibility represents a separate embodiment of the invention.
[0376] Therefore, according to another aspect of the present invention, a method is provided for producing a long dsRNA molecule in a plant cell capable of silencing a harmful organism gene, the method comprising: (a) Select a nucleic acid sequence in a plant genome that encodes a silencing molecule that targets a plant gene and is capable of recruiting RNA-dependent RNA polymerase (RdRp). (b) Modifying a nucleic acid sequence of the plant gene to impart a silencing specificity to the harmful organism gene, such that a transcript of the plant gene containing the silencing specificity forms a base complement with the silencing molecule capable of recruiting the RdRp, thereby producing the long dsRNA molecule capable of silencing the harmful organism gene. This results in the production of long dsRNA molecules in the plant cells that can silence the genes of the harmful organism.
[0377] According to one embodiment, the plant gene does not encode a molecule with an intrinsic silencing activity.
[0378] According to one embodiment, when the plant gene does not encode a molecule having an intrinsic silencing activity, the method further includes introducing a DNA editing agent into the plant cell, the DNA editing agent conferring a silencing specificity on the plant gene against a harmful organism gene.
[0379] According to one embodiment, the plant gene encodes a molecule that has an intrinsic silencing activity against a natural plant gene.
[0380] According to one embodiment, the plant gene having an intrinsic silencing activity is selected from a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a trans-acting siRNA (tasiRNA), a phased small interfering RNA (phasiRNA), a transfer RNA (tRNA), a small nuclear RNA (snRNA), a ribosomal RNA (rRNA), a small nucleolar RNA (snoRNA), an extracellular RNA (exRNA), a repetitive derived RNA, and autonomous and non-autonomous transposable RNA.
[0381] According to some embodiments, the plant gene encoding an RNA having the intrinsic silencing activity encodes a phase-dependent secondary-generation siRNA molecule.
[0382] As used herein, the phrase "phased secondary siRNA-producing molecule" refers to an RNA transcript capable of forming base complementarity with a primary silencing molecule (e.g., an miRNA), which recruits an RNA-dependent RNA polymerase (RdRp) to be transcribed into a long dsRNA molecule, which is then processed into several secondary silencing RNA molecules (i.e., several phased RNAs). According to some embodiments, the phased secondary siRNA-producing molecule is selected from a group consisting of a tasiRNA and a phasiRNA.
[0383] According to some embodiments, the phased secondary siRNA generating molecule can be processed into multiple secondary silent RNA molecules, i.e., at least two secondary silent RNA molecules. According to some embodiments, the modification step of the gene encoding the phased secondary siRNA generating molecule includes modifying only a portion of the plurality of secondary silent RNA molecules formed by processing the phased secondary siRNA generating molecule. According to a specific embodiment, the modification step of the gene encoding the phased secondary siRNA generating molecule includes modifying only one secondary silent RNA molecule formed by processing the phased secondary siRNA generating molecule. According to some embodiments, the modification step of the gene encoding the phased secondary siRNA generating molecule includes modifying at least one secondary silent RNA molecule formed by processing the phased secondary siRNA generating molecule. According to several other embodiments, the modification step of the gene encoding the phased secondary siRNA generating molecule includes modifying all secondary silent RNA molecules formed by processing the phased secondary siRNA generating molecule. Without being bound by theories or mechanisms, the gene encoding the phased secondary siRNA producing molecule was modified such that the silencing specificity of only one of the plurality of secondary silencing RNA molecules against a new target (e.g., a harmful biological RNA) is sufficient to induce at least partial silencing of the new target.
[0384] According to some embodiments, the length of the secondary silencing RNA molecule sequence to be modified is the length of the secondary silencing molecule within the targeted pest (e.g., if a tasiRN is processed within a pest to form several 24nt secondary sRNAs, then the gene sequence encoding the phased secondary siRNA-producing molecule in a plant cell is modified such that at least one 24nt sequence targets the selected pest RNA). According to some embodiments, the step of modifying a nucleic acid sequence of the plant gene (e.g., a plant gene encoding a phased secondary siRNA-producing molecule) to confer a silencing specificity against a pest gene includes modifying a sequence of 21 to 30nt, optionally 24nt, possibly 30nt in the plant gene, such that the coding sequence is substantially complementary to an RNA encoded by the pest gene. Each possibility represents a separate embodiment of the invention. Unwilling to be bound by theories or mechanisms, the gene encoding the phased secondary siRNA-producing molecule was modified such that a 30 nt portion of the coding sequence was complementary to the pest gene, ensuring (potentially different from processing within the plant gene) the processing of the long dsRNA, resulting in several secondary RNA molecules with functional silencing activity in the pest. According to a specific embodiment, the plant gene with the intrinsic silencing activity is a trans-acting siRNA (TAS)-producing molecule.
[0385] According to one specific embodiment, the plant gene includes a binding site for the silencing molecule.
[0386] According to one specific embodiment, the plant gene includes a binding site for a miRNA molecule.
[0387] According to a specific embodiment, miRNAs include, but are not limited to, miR-156a, miR-156c, miR-162a, miR-162b, miR-167d, miR-169b, miR-173, miR-393a, and miR- 393b,miR-402,miR-403,miR-447a,miR-447b,miR-447c,miR-472,miR-771,miR-777,miR-828,miR-830,miR-831,miR-831,m iR-833a,miR-833a,miR-840,miR-845b,miR-848,miR-850,miR-853,miR-855,miR-856,miR-864,miR-2933a,miR-2933b,miR- 2936,miR-4221,miR-5024,miR-5629,miR-5648,miR-5996,miR-8166,miR-8167a,miR-8167b,miR-8167c,miR-8167d,miR-87f6emiR-8177 andmiR-8182.
[0388] According to one embodiment, when the plant gene encodes a molecule having an intrinsic silencing activity, the method further includes introducing a DNA editing agent into the plant cell, the DNA editing agent specifically redirecting the silencing of the plant gene to the harmful organism gene, the harmful organism gene being different from the natural plant gene.
[0389] As used herein, the term "redirects a silencing specificity" refers to reprogramming the original specificity of the RNA molecule or the transcript of the plant gene to a non-natural target of the RNA molecule or the plant gene transcript. Thus, the original specificity of the RNA molecule or the plant gene transcript is abolished (i.e., lost function), and the new specificity is directed against a different target than the natural target (i.e., RNA of a plant or a pest, respectively), thus gaining a functional gain. It should be understood that this functional gain only occurs if the RNA molecule or the transcript of the plant gene does not possess intrinsic silencing activity.
[0390] As used herein, the term "natural plant RNA" refers to an RNA sequence naturally bound to an RNA molecule (e.g., a non-coding RNA molecule, such as a silencing molecule). Therefore, those skilled in the art will regard said natural plant RNA (i.e., a transcript of a natural plant gene) as a natural matrix (i.e., a target) of said RNA molecule (e.g., a non-coding RNA, such as a silencing molecule).
[0391] As used herein, the term "plant RNA" or "plant target RNA" refers to an RNA sequence (coding or non-coding) that is not naturally bound by an RNA molecule (e.g., a non-coding RNA, such as a silencing molecule). Therefore, the plant RNA (i.e., a transcript of a plant gene) is not a natural matrix (i.e., a target) of the RNA molecule (e.g., a non-coding RNA, such as a silencing molecule).
[0392] As used herein, the terms "pest RNA" or "pest target RNA" refer to an RNA sequence that is silenced by the plant RNA and / or several dsRNA molecules and several secondary small RNAs (generated through dsRNA processing). Therefore, the pest RNA (i.e., a transcript of a pest gene) is not a natural matrix (i.e., a target) of the plant RNA, the dsRNA, or the several secondary small molecules.
[0393] As used herein, the phrase "silencing a gene" refers to the absence or observable reduction in the levels of mRNA and / or several protein products from the target gene (e.g., due to co-transcriptional and / or post-transcriptional gene silencing). Therefore, the silencing of a target gene can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a gene not targeted by the RNA molecules designed in this invention.
[0394] The aforementioned results of silence can be confirmed by examining the aforementioned external characteristics of a plant cell or whole plant or other organism (e.g., a pest) that has absorbed the RNA designed from the plant, or by several biochemical techniques (as discussed further herein).
[0395] It should be understood that the designed RNA molecules of some embodiments of the present invention may have some off-target specificity effect, as long as it does not affect an agriculturally valuable trait (e.g., the biomass, yield, growth, etc. of the plant). The specific binding of an RNA molecule (e.g., a silencing molecule) to a target RNA can be determined by computational algorithms (e.g., BLAST) and verified by methods including, for example, Northern Imprinting, In Situ Hybridization, QuantiGene Plex Assay, etc.
[0396] According to one embodiment, if the RNA molecule is a siRNA or has been processed into a siRNA, then the complementarity with its target sequence is in the range of 90% to 100% (e.g., 100%).
[0397] According to one embodiment, if the RNA molecule is a miRNA or piRNA or has been processed into a miRNA or piRNA, then the complementarity with its target sequence is in the range of 33% to 100%.
[0398] According to one embodiment, if the RNA molecule is a miRNA, then the complementarity of the seed sequence with its target sequence (i.e., the 2nd to 8th nucleotides from the 5' end) is in the range of 85-100% (e.g., 100%).
[0399] According to one embodiment, the complementarity with the target sequence is at least about 33% (e.g., 33% of 21 to 28 nt) of the processed small RNA form. Therefore, for example, if the RNA molecule is a miRNA, then 33% of the mature miRNA sequence (e.g., 21 nt) includes seed complementarity (e.g., 7 nt of 21 nt).
[0400] According to one embodiment, the complementarity with the target sequence is at least about 45% (e.g., 45% of 21 to 28 nt) of the processed small RNA form. Thus, for example, if the RNA molecule is a miRNA, then 45% of the mature miRNA sequence (e.g., 21 nt) includes seed complementarity (e.g., 9 to 10 nt of the 21 nt).
[0401] According to one embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have about 10%, 20%, 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or up to 99% complementarity with the sequence respectively targeting the plant RNA or the harmful organism RNA.
[0402] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 99% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0403] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 98% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0404] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 97% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0405] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 96% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0406] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 95% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0407] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 94% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0408] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 93% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0409] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 92% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0410] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 91% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0411] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 90% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0412] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 85% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0413] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 50% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0414] According to one specific embodiment, the RNA molecule or plant RNA (i.e., prior to modification) is typically selected to have no more than 33% complementarity with the sequence of the plant RNA or the harmful organism RNA, respectively.
[0415] According to one embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least about 33%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity of the sequence against the plant RNA or the harmful organism RNA, respectively.
[0416] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 33% complementarity (e.g., 85% to 100% seed match) against the plant RNA or the harmful organism RNA, respectively.
[0417] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 40% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0418] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 45% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0419] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 50% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0420] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 55% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0421] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 60% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0422] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 70% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0423] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 80% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0424] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 85% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0425] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 90% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0426] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 91% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0427] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 92% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0428] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 93% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0429] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 94% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0430] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 95% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0431] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 96% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0432] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 97% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0433] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 98% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0434] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include at least 99% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0435] According to one specific embodiment, the RNA molecule (e.g., an RNA silencing molecule) or plant RNA is designed to include 100% complementarity against the plant RNA or the harmful organism RNA, respectively.
[0436] According to a specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least about 33%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity to the sequence targeting the harmful organism RNA.
[0437] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include a minimum of 33% complementarity (e.g., 85 to 100% seed match) of the sequence against the harmful organism RNA.
[0438] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 40% complementarity to the sequence of the harmful organism RNA.
[0439] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 45% complementarity to the sequence of the harmful organism RNA.
[0440] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 50% complementarity to the sequence of the harmful organism RNA.
[0441] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 55% complementarity to the sequence targeting the harmful organism RNA.
[0442] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 60% complementarity to the sequence of the harmful organism RNA.
[0443] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 70% complementarity to the sequence of the harmful organism RNA.
[0444] According to a specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 80% complementarity to the sequence of the harmful organism RNA.
[0445] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 85% complementarity to the sequence targeting the harmful organism RNA.
[0446] According to a specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 90% complementarity to the sequence of the harmful organism RNA.
[0447] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 91% complementarity to the sequence of the harmful organism RNA.
[0448] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 92% complementarity to the sequence targeting the harmful organism RNA.
[0449] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 93% complementarity to the sequence of the harmful organism RNA.
[0450] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 94% complementarity to the sequence targeting the harmful organism RNA.
[0451] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 95% complementarity to the sequence of the harmful organism RNA.
[0452] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 96% complementarity to the sequence targeting the harmful organism RNA.
[0453] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 97% complementarity to the sequence targeting the harmful organism RNA.
[0454] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 98% complementarity to the sequence of the harmful organism RNA.
[0455] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include at least 99% complementarity to the sequence of the harmful organism RNA.
[0456] According to one specific embodiment, the antisense strand of the RNA molecule or plant RNA (e.g., a product synthesized from RdRp) is designed to include 100% complementarity to the sequence targeting the harmful organism RNA.
[0457] To induce silencing activity and / or specificity of an RNA molecule or a plant RNA, or to redirect the silencing activity and / or specificity of an RNA molecule or a plant RNA (e.g., an RNA silencing molecule) to a plant RNA or a harmful organism RNA, a DNA editing agent is used to modify the gene encoding an RNA molecule or said plant RNA (e.g., an RNA silencing molecule).
[0458] The following is a description of various non-limiting examples of methods and DNA editing agents for introducing several nucleic acid alterations into a gene, as well as several reagents for carrying out the methods and DNA editing agents that can be used according to several specific embodiments of this disclosure.
[0459] Genome editing using engineered nucleases is a reverse genetics approach that typically uses engineered nucleases to cut at several desired locations in the genome, creating several specific double-stranded breaks (DSBs), which are then repaired by several endogenous cellular processes, such as homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends with a single DSB (with or without minimal end modification), while HR utilizes a homologous donor sequence as a template (i.e., sister chromatids formed in S phase) to regenerate / replicate the missing DNA sequence at the break site. For several specific nucleotide modifications to be introduced into the genomic DNA, a donor DNA repair template (exogenously provided single-stranded or double-stranded DNA) containing the desired sequence must be present during HR.
[0460] Genome editing cannot be performed using traditional restriction endonucleases because most restriction enzymes target only a few base pairs on the DNA, and these sequences are typically found in many locations across the genome, resulting in multiple cuts that are not limited to a single desired location. To overcome this challenge and generate several site-specific single- or double-strand breaks (DSBs), several different classes of nucleases have been discovered and bioengineered to date. These include meganucleases, zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and the CRISPR / Cas9 system.
[0461] Meganucleases—also known as homing endonucleases—are generally classified into at least five families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, the HNH family, and the PD-(D / E)xK family. These at least five families involve several EDxHD enzymes and are considered by some to be a single family. These families are characterized by several structural motifs that influence catalytic activity and recognition sequences. For example, several members of the LAGLIDADG family are characterized by having one or two copies of a preserved LAGLIDADG motif. These four families of meganucleases are highly differentiated from each other in terms of several preserved structural elements, DNA recognition sequence specificity, and catalytic activity. Meganucleases are typically found in microbial species and possess a unique characteristic of having several very long recognition sequences (>14 bp), thus naturally making them highly specific for cleavage at a desired location.
[0462] This can be used to create several site-specific double-strand breaks (DSBs) in genome editing. Those skilled in the art can use these naturally occurring broad-spectrum nucleases, but the number of such naturally occurring broad-spectrum nucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate several broad-spectrum nuclease variants that recognize several unique sequences. For example, various broad-spectrum nucleases have been fused to generate several hybrid enzymes that recognize a new sequence.
[0463] Alternatively, several DNA-interacting amino acids of the said macronuclease can be altered to design sequence-specific macronucleases (e.g., see U.S. Patent No. 8,021,867). Macronucleases can be designed using, for example, methods described in Certo, MT et al. (Nature Methods, 2012, 9:073-975), U.S. Patent Nos. 8,304,222, 8,021,867, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015, 8,143,016, 8,148,098, or 8,163,514, each of which is incorporated herein by reference in its entirety. Alternatively, commercially available technologies (e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology) can be used to obtain macronucleases with site-specific cleavage characteristics.
[0464] ZFNs and TALENs – two different engineered nucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both been shown to efficiently generate several targeted double-strand breaks (DSBs) (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).
[0465] Essentially, ZFN and TALEN restriction endonuclease technologies utilize a non-specific DNA-cutting enzyme linked to a specific DNA-binding domain (either a series of zinc finger domains or several TALE repeat sequences). Typically, a restriction enzyme is chosen whose DNA recognition site and cleavage site are separated from each other. Separating the cleavage site and then linking it to a DNA-binding domain produces a nuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with this property is Fok1. Furthermore, an advantage of Fok1 is that dimerization is required to acquire nuclease activity, meaning that the specificity increases significantly as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, several Fok1 nucleases have been engineered to function only as heterodimers and possess increased catalytic activity. These heterodimers prevent the nuclease from exhibiting the potential for unwanted homodimer activity, thus increasing the specificity for double-strand breaks (DSBs).
[0466] Therefore, for example, to target a specific site, several ZFNs and several TALENs are constructed as several nuclease pairs, each molecule in the pair being designed to bind to several adjacent sequences at the target site. After transient expression in several cells, the several nucleases bind to their several target sites, and the several FokI domains heterodimerize to produce a double-strand break (DSB). Repair of these double-strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway typically results in several small deletions or several small sequence insertions (indels). Since each repair via NHEJ is unique, a single nuclease pair can generate an allelic series with a series of several different insertions or deletions at the target site.
[0467] Generally, NHEJ is relatively accurate in gene editing (approximately 85% of DSBs in human cells are repaired by NHEJ within about 30 minutes of detection). Incorrect NHEJ is dependent because when the repair is accurate, the nuclease will continue to cleave until the repair product is mutated, and the recognition / cleavage site / PAM motif is lost / mutated, or the transiently introduced nuclease is no longer present.
[0468] The length of deletions typically ranges from a few base pairs to hundreds of base pairs, but larger deletions have been successfully generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). Furthermore, when a DNA fragment homologous to the targeted region is introduced and bound to the nuclease pair, the double-strand break (DSB) can be repaired via homologous recombination (HR) to generate multiple specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).
[0469] Although multiple nuclease regions of multiple ZFNs and multiple TALENs share similar properties, the differences between these engineered nucleases lie in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains are characterized by their natural presence within their protein combinations. Multiple Cys2-His2 zinc fingers are typically found in multiple repeating sequences spaced 3 bp apart and in multiple different combinations of various nucleic acid-interacting proteins. On the other hand, multiple TALEs are found in multiple repeating sequences with a one-to-one recognition rate between the multiple amino acids and the multiple recognized nucleotide pairs. Because both zinc fingers and TALEs occur in a repeating pattern, different combinations can be attempted to create a wide variety of sequence specificities. Several methods exist for preparing site-specific zinc finger endonucleases, including, for example, modular assembly (where multiple zinc fingers associated with a triplet sequence are linked together in a row to cover the desired sequence), OPEN (low-strength selection of multiple peptide domains relative to multiple triplet nucleotides, followed by high-strength selection of multiple peptide combinations relative to the final target in multiple bacterial systems), and one-hybrid screening of multiple zinc finger libraries. ZFNs are also commercially designed and available from companies such as Sangamo Biosciences™ (Richmond, California).
[0470] Methods for designing and obtaining multiple TALENs have been described, for example, by Reyon et al. (Nature Biotechnology, May 2012; 30(5): 460-5), Miller et al. (Nat Biotechnol., 2011, 29: 143-148), Cermak et al. (Nucleic Acids Research, 2011, 39(12): e82), and Zhang et al. (Nature Biotechnology, 2011, 29(2): 149-53). A recently developed web-based program (called the Mojo Hand), introduced by the Mayo Clinic, is used to design multiple TAL and TALEN constructs for genome editing applications (accessible via www(dot)talendesign(dot)org). TALENs can also be commercially designed and obtained from, for example, Sangamo Biosciences™ (Richmond, CA).
[0471] The T-GEE system (TargetGene's Genome Editing Engine) provides a programmable nucleoprotein molecular complex containing a polypeptide moiety and a specificity-conferring nucleic acid (SCNA) that is assembled in vivo in a target cell and capable of interacting with a predetermined target nucleic acid sequence. The programmable nucleoprotein molecular complex can specifically modify and / or edit a target site within the target nucleic acid sequence and / or modify the function of the target nucleic acid sequence. The nucleoprotein composition includes (a) a polynucleotide molecule encoding a chimeric polypeptide and includes (i) a functional domain capable of modifying the target site, and (ii) a linker domain capable of interacting with the specificity-conferring nucleic acid; and (b) a specificity-conferring nucleic acid (SCNA) including (i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site, and (ii) a recognition region capable of specifically attaching to the linker domain of the polypeptide. The composition, by endowing specific nucleic acids with base pairing with a target nucleic acid, exhibits high specificity and binding ability of the molecular complex to the target nucleic acid, enabling accurate, reliable, and cost-effective modification of a predetermined nucleic acid sequence target. The composition also features low genotoxicity, modular assembly, utilization of a single, custom-designed platform, independent use outside of dedicated core facilities, and short development cycles with low cost.
[0472] The CRISPR-Cas system and all its variants (also referred to herein as “CRISPR”) – many bacteria and archaea possess an adaptive immune system based on endogenous RNA that degrades multiple nucleic acids that invade multiple bacteriophages and plasmids. These systems consist of multiple clustered regularly interspaced short palindromic repeat (CRISPR) nucleotide sequences that produce multiple RNA components and multiple CRISPR-associated (Cas) genes (encoding multiple protein components). These multiple CRISPR RNAs (crRNAs) contain short homologs to the DNA of multiple specific viruses and plasmids and guide Cas nucleases to degrade multiple nucleic acids complementary to the corresponding pathogens. Studies of the type II CRISPR / Cas system for Streptococcus pyogenes indicate that three components form an RNA / protein complex that together are sufficient to satisfy sequence-specific nuclease activity: the Cas9 nuclease, a crRNA homologous to the target sequence by 20 base pairs, and a trans-activating crRNA (tracrRNA) (Jinek et al., Science, 2012, 337: 816-821).
[0473] Further, it was demonstrated that a synthetic chimeric guide RNA (gRNA) consisting of a fusion between crRNA and tracrRNA can direct Cas9 in vitro to cleave multiple DNA targets complementary to the crRNA. It was also demonstrated that co-transient expression of Cas9 with multiple synthetic gRNAs can be used to generate targeted multiple double-strand breaks (DSBs) in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a, b; Jinek et al., 2013; Mali et al., 2013).
[0474] The CRISPR / Cas system used for genome editing contains two distinct components: an sgRNA and an endonuclease, such as Cas9.
[0475] The gRNA (also referred to herein as short guide RNA (sgRNA)) is typically a 20-nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA, which is linked to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The sgRNA / Cas9 complex is recruited to the target sequence via base pairing between the sgRNA sequence and the complement genomic DNA. For successful Cas9 binding, the genomic target sequence must also contain the correct protospacer adjacent motif (PAM) sequence immediately following the target sequence. The binding of the sgRNA / Cas9 complex positions the Cas9 onto the genomic target sequence, allowing the Cas9 to cleave both strands of the DNA, resulting in a double-strand break (DSB). Similar to several ZFNs and several TALENs, these double-strand breaks (DSBs) generated by CRISPR / Cas are susceptible to homologous recombination or NHEJ and are prone to specific sequence modifications during DNA repair.
[0476] The Cas9 nuclease has two functional domains: RuvC and HNH, each cleaving a different DNA strand. When both domains are active, Cas9 induces multiple double-strand breaks (DSBs) in the genomic DNA.
[0477] A significant advantage of CRISPR / Cas is the combination of its high efficiency and its ability to readily generate multiple synthetic gRNAs. This results in a system that can be easily modified to target multiple modifications at different genomic sites and / or multiple different modifications at the same site. Furthermore, protocols capable of simultaneously targeting multiple genes have been established. Most cells carrying these mutations exhibit multiple biallelic mutations in the targeted genes.
[0478] However, the apparent flexibility in the base pairing interactions between the sgRNA sequence and the genomic DNA target sequence allows for incomplete matching to the target sequence that will be cleaved by Cas9.
[0479] Multiple modified forms of the Cas9 enzyme containing a single inactive catalytic domain (RuvC- or HNH-) are called "nickases." Having only one active nuclease domain, a Cas9 nickase cleaves only one strand of the target DNA, resulting in a single-strand break or "nick." A single-strand break or nick is mostly repaired by single-strand break repair mechanisms involving proteins, such as, but not limited to, PARP (sensor) and the XRCC1 / LIG III complex (ligation). If a single-strand break (SSB) is generated by multiple topoisomerase I poisons or by multiple drugs capturing RARP1 on a naturally occurring SSB, these may persist and become multiple single-ended DSBs when the cell enters S phase and the replication fork encounters such SSBs, which can only be repaired by HR. However, two proximal, opposite strand cuts introduced by a Cas9 nick are considered a double-strand break, typically found in a CRISPR system known as a "double nick." A double nick, essentially a non-parallel DSB, can be repaired by HR or NHEJ, like other DSBs, depending on the desired action on the gene target, the presence of a donor sequence, and the cell cycle stage (HR is less abundant and occurs only in the S and G2 phases of the cell cycle). Therefore, if specificity and reduced off-target effects are critical, using the Cas9 nick to generate a double nick (by designing two sgRNAs and multiple target sequences adjacent to each other on multiple opposing strands of the genomic DNA) will reduce off-target effects, as either sgRNA alone would create a nick that is unlikely to alter the genomic DNA, even if such events are not impossible.
[0480] The modified form of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9 or dCas9) lacks nuclease activity but can still bind to DNA based on sgRNA specificity. The dCas9 can serve as a platform for multiple DNA transcription regulators, activating or repressing gene expression by fusing the inactive enzyme to multiple known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
[0481] Several other variants of Cas9 may be used by some embodiments of the present invention, including but not limited to CasX and Cpf1. Several CasX enzymes comprise a distinct family of RNA-guided genome editing agents that are smaller in size and present in bacteria (typically absent in humans) than Cas9, and are therefore less likely to elicit a human immune system / response. Furthermore, CasX uses a different PAM motif than Cas9, and can therefore be used to target several sequences for which several Cas9 PAM motifs have not been found [see Liu JJ et al., Nature. (2019) 566(7743): 218-223.]. Cpf1 (also known as Cas12a) is particularly advantageous for editing several AT-rich regions with much lower Cas9 PAM (NGG) content [see Li T et al., Biotechnol Adv. (2019) 37(1): 21-27; Murugan K et al., Mol Cell. (2017) 68(1): 15-25].
[0482] According to another embodiment, the CRISPR system can be fused with various effector domains, such as a DNA cleavage domain. The DNA cleavage domain can be obtained from any endonuclease or exonuclease. Several non-limiting examples of endonucleases from which a DNA cleavage domain can be derived include, but are not limited to, several restriction endonucleases and several homing endonucleases (see, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.). In several exemplary embodiments, the cleavage domain of the CRISPR system is a Fok1 endonuclease domain or a modified Fok1 endonuclease domain. Furthermore, using several homing endonucleases (HE) is another option. HE are several small proteins (<300 amino acids) found in bacteria, archaea, and single-celled eukaryotes. A notable characteristic of HE is that, compared to other site-specific endonucleases (e.g., restriction endonucleases (4 to 8 bp)), they recognize relatively long sequences (14 to 40 bp). HE has historically been classified as several small conserved amino acid motifs. At least five such families have been identified: LAGLIDADG; GIY-YIG; HNH; His-Cys Box; and PD-(D / E)xK, which are associated with several EDxHD enzymes and are considered by some to be a separate family. At a structural level, the HNH and His-Cys Box share a common fold (designated as ββα-metal) with the PD-(D / E)xK and several EDxHD enzymes. Each family has different catalytic and DNA recognition strategies and is suited to various engineering applications to varying degrees. See, for example, Methods Mol Biol., (2014) 1123:1-26. Exemplary homing endonucleases that may be used in some embodiments of the present invention include, but are not limited to, I-CreI, I-TevI, I-HmuI, I-PpoI, and I-Ssp68031.
[0483] Several modified versions of CRISPR, such as dead CRISPR (dCRISPR-endonuclease), can also be used for CRISPR transcriptional repression (CRISPRi) or CRISPR transcriptional activation (CRISPRa), see, for example, Kampmann M., ACSChem Biol., (2018) 13(2): 406-416; La Russa MF and Qi LS., Mol Cell Biol. (2015) 35(22): 3800-9.
[0484] Several other versions of CRISPR can be used according to some embodiments of the present invention, including genome editing, which uses several components from several CRISPR systems, along with several other enzymes, to directly install several point mutations in cellular DNA or RNA.
[0485] Therefore, according to one embodiment, the editing agent is a DNA or RNA editing agent.
[0486] According to one embodiment, the DNA or RNA editing agent induces base editing.
[0487] The term "base editing" as used in this article refers to the insertion of several point mutations into cellular DNA or RNA without causing double-strand or single-strand DNA breaks.
[0488] In base editing, several DNA base editors typically comprise a fusion between a catalytically impaired Cas nuclease and a base-modifying enzyme that acts on single-stranded DNA (ssDNA). Upon binding to its target DNA site, base pairing between the gRNA and the target DNA strand results in the substitution of a small segment of single-stranded DNA within an "R loop." The DNA bases within the ssDNA bubble are modified by the base-editing enzyme (e.g., a deaminase). To improve efficiency across multiple eukaryotic cells, the catalytically impaired Cas nuclease also creates a cut in the unedited DNA strand, inducing multiple cells to use the edited strand as a template to repair the unedited strand.
[0489] Two classes of DNA base editors have been described: several cytosine base editors (CBEs) convert a CG base pair to a TA base pair, and several adenine base editors (ABEs) convert an AT base pair to a GC base pair. In general, several CBEs and several ABEs can mediate all four possible transition mutations (C to T, A to G, T to C, and G to A). Similarly, in RNA, the conversion of targeted adenosine to inosine is achieved through several methods that simultaneously utilize antisense and Cas13-guided RNA targeting.
[0490] According to one embodiment, the DNA or RNA editing agent includes a catalytically inactive endonuclease (e.g., CRISPR-dCas).
[0491] According to one embodiment, the catalytically inactivated endonuclease is an inactive Cas9 (e.g., dCas9).
[0492] According to one embodiment, the catalytically inactivated endonuclease is an inactive Cas13 (e.g., dCas13).
[0493] According to one embodiment, the DNA or RNA editing agent includes an enzyme capable of performing epigenetic editing (i.e., providing several chemical changes to the DNA, RNA, or histone).
[0494] Several exemplary enzymes include, but are not limited to, several DNA methyltransferases, several methyltransferases, and several acetyltransferases. More specifically, several exemplary enzymes include, for example, DNA (cytosine-5)-methyltransferase 3A (DNA (DNMT3a), histone acetyltransferase p300, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), lysine (K)-specific demethylase 1A (LSD1), and calcium and integrin binding protein 1 (CIB1).
[0495] In addition to the catalytically disabled nuclease, the various DNA or RNA editing agents of the present invention may also include a nucleobase deaminase and / or a DNA glycosylation inhibitor.
[0496] According to a specific embodiment, the DNA or RNA editing agent comprises BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), or BE3 (APOBEC-XTEN-dCas9(A840H)-UGI), and includes sgRNA. APOBEC1 is a full-length or catalytically active deaminase fragment, XTEN is a protein linker, UGI is a uracil DNA glycosylase inhibitor to prevent subsequent U:G mismatches from being repaired back to a C:G base pair, and dCas9 (A840H) is an absorptive enzyme, wherein the dCas9 is reduced to restore the catalytic activity of the HNH domain, which cleaves only the unedited strand, mimicking newly synthesized DNA and producing the desired U:A product.
[0497] According to some embodiments of the present invention, other enzymes that can be used for base editing are described in detail in the following literature, namely Rees and Liu, Nature Reviews Genetics (2018) 19:770 to 788, which are incorporated herein by reference in their entirety.
[0498] There are many publicly available tools to help select and / or design multiple target sequences, as well as multiple unique sgRNA lists for different genes in different species identified by bioinformatics, such as, but not limited to, Target Finder from Feng Zhang’s lab, E-CRISP from Michael Boutros’ lab, RGEN tools: Cas-OFFinder, CasFinder: a flexible algorithm for identifying specific Cas9 targets in the genome, and CRISPR Optimal Target Finder.
[0499] For use of the CRISPR system, both the sgRNA and a Cas endonuclease (e.g., Cas9) should be expressed or present in a target cell (e.g., as a ribonucleoprotein complex). The insert vector may contain two cassettes on a single plasmid, or the multiple cassettes may be expressed by two separate plasmids. Multiple CRISPR plasmids are commercially available, such as the px330 plasmid from Addgene (75 Sidney St, Suite 550A, Cambridge, MA 02139). The use of clustered regularly spaced short palindromic repeats (CRISPR) associated (Cas) guide RNA technology and Cas endonuclease modification of plant genomes is disclosed at least in Svitashev et al. (2015, Plant Physiology, 169(2): 931-945), Kumar and Jain (2015, J Exp Bot, 66: 47-57), and in U.S. Patent Application Publication No. 20150082478, the contents of which are incorporated herein by reference in their entirety. Cas endonucleases that can be used for DNA editing via gRNA include, but are not limited to, Cas9, Cpfl (Zetsche et al., 2015, Cell, 163(3): 759-71), C2c1, C2c2, and C2c3 (Shmakov et al., Mol Cell., 5 Nov 2015; 60(3): 385-97).
[0500] The “hit-and-run” or “in-out” gene targeting strategy involves a one- or two-step recombination procedure. In the first step, an insert vector containing a dual positive / negative selection cassette is used to introduce the desired sequence change. The insert vector contains a single, contiguous region homologous to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a site within the homologous region, introduced into the plurality of cells, and positively selected to isolate homologous recombination-mediated events. The DNA carrying the homologous sequence can be provided as a plasmid, single-stranded, or double-stranded oligonucleotide. These homologous recombinants contain a localized replication separated by an intermediate vector sequence containing the selection cassette. In the second step, negative selection is performed on the plurality of targeted clones to identify the plurality of cells that have lost the selection cassette through intrachromosomal recombination between the repetitive sequences. The localized recombination event removes the repetition, and depending on the recombination site, the allele retains the introduced mutation or reverts to the wild type. The end result is that the desired modifications are introduced without preserving any exogenous sequences.
[0501] The "double-replacement" or "tag and exchange" strategy involves a one- or two-step selection procedure, similar to the hit-and-run gene targeting strategy, but requiring the use of two different target constructs. In the first step, a standard targeting vector with 3' and 5' homologous arms is inserted near the site where the mutation will be introduced, creating a dual positive / negative selection cassette. Introducing this system component into the cell and performing positive selection identifies HR-mediated events. Next, a second targeting vector containing a homologous region to the desired mutation is introduced into multiple target clones, and negative selection is performed to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.
[0502] According to one specific embodiment, the DNA editing agent includes a DNA targeting module (e.g., sgRNA).
[0503] According to one specific embodiment, the DNA editing agent does not include a nuclease.
[0504] According to one specific embodiment, the DNA editing agent includes a nuclease (e.g., an endonuclease) and a DNA targeting module (e.g., sgRNA).
[0505] According to one specific embodiment, the DNA editing agent is CRISPR / Cas, such as sgRNA and Cas9.
[0506] According to one specific embodiment, the DNA editing agent is TALEN.
[0507] According to one specific embodiment, the DNA editing agent is ZFN.
[0508] According to one specific embodiment, the DNA editing agent is a broad-spectrum nuclease.
[0509] According to one specific embodiment, the DNA editing agent includes a CRISPR endonuclease and an sgRNA directed to cleave the plant gene.
[0510] According to one specific embodiment, an oligonucleotide serving as a template for homology-dependent recombination (HDR) is introduced into the cell along with the DNA editing agent, wherein the oligonucleotide comprises a sequence of the plant gene having several nucleotide alterations that modify the nucleic acid sequence of the plant gene to impart a silencing specificity against the harmful organism gene.
[0511] According to one embodiment, the DNA editing agent is linked to a reporter expressed in a plant cell.
[0512] According to one embodiment, the reporter is a fluorescent reporter protein.
[0513] The term "fluorescent protein" refers to a polypeptide that emits fluorescence and is typically detected by flow cytometry, microscopy, or any fluorescence imaging system, and can therefore serve as a basis for selecting multiple cells that express this protein.
[0514] Examples of fluorescent proteins that can be used as multiple reporters include, but are not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent proteins (e.g., dsRed, mCherry, RFP). A non-limiting list of fluorescent or other reporters includes multiple proteins that can be detected by luminescence (e.g., luciferase) or colorimetric assays (e.g., GUS). According to a specific embodiment, the fluorescent reporter is a red fluorescent protein (e.g., dsRed, mCherry, RFP) or GFP.
[0515] A review of multiple new classes and applications of various fluorescent proteins can be found in Trends in Biochemical Sciences (Rodriguez, Erik A.; Campbell, Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, Amy E.; Shu, Xiaokun; Zhang, Jin; Tsien, Roger Y., “The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins”, Trends in Biochemical Sciences, doi: 10.1016 / j.tibs.2016.09.010).
[0516] According to another embodiment, the reporter is an endogenous gene of a plant. An exemplary reporter is the phytoene desaturase gene (PDS3), which encodes one of several important enzymes in the carotenoid biosynthesis pathway; its silencing produces an albino / bleached phenotype. Therefore, several plants with reduced PDS3 expression exhibit reduced chlorophyll levels, up to complete albinism and dwarfism. Other genes that may be used according to this teaching include, but are not limited to, several genes involved in crop protection. Several exemplary genes are described in Table 1B below.
[0517] According to another embodiment, the reporter is an antibiotic selection marker. Examples of multiple antibiotic selection markers that can be used as multiple reporter are, but are not limited to, neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt). Other marker genes that may be used according to this teaching include, but are not limited to, gentamicin acetyltransferase (accC3) resistance and bleomycin and phleomycin resistance genes.
[0518] It should be understood that the enzyme NPTII inactivates many aminoglycoside antibiotics, such as kanamycin, neomycin, geneticin (or G418), and paromomycin, through phosphorylation. Among these, kanamycin, neomycin, and paromomycin are used in a variety of plant species.
[0519] According to another embodiment, the reporter is a toxicity selection marker. An exemplary toxicity selection marker that can be used as a reporter is, but is not limited to, allyl alcohol selection using the alcohol dehydrogenase (ADH1) gene. ADH1 comprises a group of dehydrogenases that catalyze the interconversion between multiple alcohols and multiple aldehydes or multiple ketones, as well as the simultaneous reduction and degradation of multiple alcoholic toxic substances in multiple tissues by NAD+ or NADP+. Several plants with reduced ADH1 expression exhibit increased tolerance to allyl alcohol. Therefore, several plants with reduced ADH1 are resistant to the toxic effects of allyl alcohol.
[0520] Regardless of the DNA editing agent used, the method of the present invention is employed such that the gene encoding the RNA molecule or the plant gene (e.g., an RNA silencing molecule) is modified by at least one of a deletion, an insertion, or a point mutation.
[0521] According to one embodiment, the modification is in a structured region of the non-coding RNA molecule (e.g., an RNA silencing molecule).
[0522] According to one embodiment, the modification is in a stem region of a non-coding RNA molecule (e.g., an RNA silencing molecule).
[0523] According to one embodiment, the modification is in a loop region of a non-coding RNA molecule (e.g., an RNA silencing molecule).
[0524] According to one embodiment, the modification is performed in a stem region and a loop region of a non-coding RNA molecule (e.g., an RNA silencing molecule).
[0525] According to one embodiment, the modification is in an unstructured region of a non-coding RNA molecule (e.g., an RNA silencing molecule).
[0526] According to one embodiment, the modification is performed on a stem region, a loop region, and an unstructured region of a non-coding RNA molecule (e.g., an RNA silencing molecule).
[0527] According to one specific embodiment, the modification includes a modification of about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 1 to 50 nucleotides, about 1 to 10 nucleotides, about 50 to 150 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0528] According to one embodiment, the modification comprises a modification of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or up to 250 nucleotides (compared to natural plant RNA or natural RNA molecules (e.g., RNA silencing molecules)).
[0529] According to one embodiment, the modification can be in a continuous nucleic acid sequence (e.g., at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 bases).
[0530] According to one embodiment, the modification may be in a non-continuous manner, for example, across a nucleic acid sequence of 20, 50, 100, 150, 200, 500, or 1000.
[0531] According to one specific embodiment, the modification includes a modification of up to 200 nucleotides.
[0532] According to one specific embodiment, the modification includes a modification of up to 150 nucleotides.
[0533] According to one specific embodiment, the modification includes a modification of up to 100 nucleotides.
[0534] According to one specific embodiment, the modification includes a modification of up to 50 nucleotides.
[0535] According to one specific embodiment, the modification includes a modification of up to 25 nucleotides.
[0536] According to one specific embodiment, the modification includes a modification of up to 20 nucleotides.
[0537] According to one specific embodiment, the modification includes a modification of up to 15 nucleotides.
[0538] According to one specific embodiment, the modification includes a modification of up to 10 nucleotides.
[0539] According to one specific embodiment, the modification includes a modification of up to 5 nucleotides.
[0540] According to one embodiment, the modification depends on the structure of the RNA molecule (e.g., a silencing molecule).
[0541] Therefore, when the RNA silencing molecule contains a non-essential structure (i.e., the primary and secondary structures of the RNA silencing molecule do not play a role in its appropriate biogenesis and / or function), or is pure dsRNA (i.e., the RNA silencing molecule has a complete or nearly complete dsRNA), some modifications (e.g., 20 to 30 nucleotides, e.g., 1 to 10 nucleotides, e.g., 5 nucleotides) are introduced to redirect the silencing specificity of the RNA silencing molecule.
[0542] According to another embodiment, when the RNA silencing molecule has an essential structure (i.e., the appropriate biogenesis and / or activity of the RNA silencing molecule depends on its secondary structure), multiple large modifications (e.g., 10 to 200 nucleotides, e.g., 50 to 150 nucleotides, e.g., more than 30 nucleotides but not more than 200 nucleotides, 30 to 200 nucleotides, 35 to 200 nucleotides, 35 to 150 nucleotides, 35 to 100 nucleotides) are introduced to redirect the silencing specificity of the RNA silencing molecule.
[0543] According to one embodiment, the modification causes the recognition / cleavage site / PAM motif of the RNA silencing molecule to be modified to eliminate the original PAM recognition site.
[0544] According to a specific embodiment, the modification is in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids in a PAM motif.
[0545] According to one embodiment, the modification includes an insertion.
[0546] According to one specific embodiment, the insertion comprises an insertion of about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 1 to 50 nucleotides, about 1 to 10 nucleotides, about 50 to 150 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0547] According to one embodiment, the insertion comprises a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or up to 250 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0548] According to one specific embodiment, the insertion includes an insertion of up to 200 nucleotides.
[0549] According to one specific embodiment, the insertion includes an insertion of up to 150 nucleotides.
[0550] According to one specific embodiment, the insertion includes an insertion of up to 100 nucleotides.
[0551] According to one specific embodiment, the insertion includes an insertion of up to 50 nucleotides.
[0552] According to one specific embodiment, the insertion includes an insertion of up to 25 nucleotides.
[0553] According to one specific embodiment, the insertion includes an insertion of up to 20 nucleotides.
[0554] According to one specific embodiment, the insertion includes an insertion of up to 15 nucleotides.
[0555] According to one specific embodiment, the insertion includes an insertion of up to 10 nucleotides.
[0556] According to one specific embodiment, the insertion includes an insertion of up to 5 nucleotides.
[0557] According to one embodiment, the modification includes a deletion.
[0558] According to one specific embodiment, the deletion includes about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 1 to 50 nucleotides, about 1 to 10 nucleotides, about 50 to 150 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0559] According to one embodiment, the deletion includes a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or up to 250 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0560] According to one specific embodiment, the deletion includes a deletion of up to 200 nucleotides.
[0561] According to one specific embodiment, the deletion includes a deletion of up to 150 nucleotides.
[0562] According to one specific embodiment, the deletion includes a deletion of up to 100 nucleotides.
[0563] According to one specific embodiment, the deletion includes a deletion of up to 50 nucleotides.
[0564] According to one specific embodiment, the deletion includes a deletion of up to 25 nucleotides.
[0565] According to one specific embodiment, the deletion includes a deletion of up to 20 nucleotides.
[0566] According to one specific embodiment, the deletion includes a deletion of up to 15 nucleotides.
[0567] According to one specific embodiment, the deletion includes a deletion of up to 10 nucleotides.
[0568] According to one specific embodiment, the deletion includes a deletion of up to 5 nucleotides.
[0569] According to one embodiment, the modification includes a point mutation.
[0570] According to one specific embodiment, the point mutation comprises about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 1 to 50 nucleotides, about 1 to 10 nucleotides, about 50 to 150 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0571] According to one embodiment, the point mutation includes a point mutation of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or up to 250 nucleotides (compared to natural plant RNA or natural RNA molecules (e.g., RNA silencing molecules)).
[0572] According to one specific embodiment, the point mutation includes a point mutation of up to 200 nucleotides.
[0573] According to one specific embodiment, the point mutation includes a point mutation of up to 150 nucleotides.
[0574] According to one specific embodiment, the point mutation includes a point mutation of up to 100 nucleotides.
[0575] According to one specific embodiment, the point mutation includes a point mutation of up to 50 nucleotides.
[0576] According to one specific embodiment, the point mutation includes a point mutation of up to 25 nucleotides.
[0577] According to one specific embodiment, the point mutation includes a point mutation of up to 20 nucleotides.
[0578] According to one specific embodiment, the point mutation includes a point mutation of up to 15 nucleotides.
[0579] According to one specific embodiment, the point mutation includes a point mutation of up to 10 nucleotides.
[0580] According to one specific embodiment, the point mutation includes a point mutation of up to 5 nucleotides.
[0581] According to one embodiment, the modification includes any combination of a deletion, an insertion, and / or a point mutation.
[0582] According to one embodiment, the modification includes nucleotide replacement (e.g., nucleotide swapping).
[0583] According to one specific embodiment, the swapping includes about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 1 to 50 nucleotides, about 1 to 10 nucleotides, about 50 to 150 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0584] According to one embodiment, the nucleotide swap comprises a nucleotide substitution of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or up to 250 nucleotides (compared to the natural plant RNA or natural RNA molecule (e.g., an RNA silencing molecule)).
[0585] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 200 nucleotides.
[0586] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 150 nucleotides.
[0587] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 100 nucleotides.
[0588] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 50 nucleotides.
[0589] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 25 nucleotides.
[0590] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 20 nucleotides.
[0591] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 15 nucleotides.
[0592] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 10 nucleotides.
[0593] According to one specific embodiment, the nucleotide exchange includes a nucleotide substitution of up to 5 nucleotides.
[0594] According to one embodiment, the gene encoding the plant RNA or RNA molecule (e.g., an RNA silencing molecule) is modified by a sequence of an endogenous RNA silencing molecule (e.g., miRNA) exchanged with a selected RNA silencing sequence (e.g., siRNA).
[0595] According to one embodiment, the guide strand of the RNA molecule (e.g., an RNA silencing molecule, such as several miRNA precursors (several pri / pre-miRNAs) or several siRNA precursors (dsRNAs)) is modified to maintain structural originality and retain the same base pairing characteristics.
[0596] According to one embodiment, the passenger strand of the RNA molecule (e.g., an RNA silencing molecule, such as several miRNA precursors (pri / pre-miRNA) or several siRNA precursors (dsRNA)) is modified to maintain structural originality and retain the same base pairing characteristics.
[0597] As used herein, the term “structural originality” refers to the secondary RNA structure (i.e., the base pairing profile). Maintaining structural originality is crucial for the proper and efficient biogenesis / processing of non-coding RNAs (e.g., RNA silencing molecules such as siRNA or miRNA), which are structure-dependent rather than purely sequence-dependent.
[0598] According to one embodiment, the RNA (e.g., an RNA silencing molecule) is modified in the guide strand (silencing strand) to include about 50 to 100% complementarity with the target RNA (as described above), while the passenger strand is modified to retain the original (unmodified) RNA (e.g., non-coding RNA) structure.
[0599] According to one embodiment, the RNA sequence (e.g., an RNA silencing molecule) is modified such that the seed sequence (e.g., 2 to 8 miRNA nucleotides starting from the 5' end) is complementary to the target sequence.
[0600] According to one specific embodiment, the RNA silencing molecule (i.e., RNAi molecule) is designed such that a sequence of the RNAi molecule is modified to maintain the originality of its structure and to be recognized by several cellular RNAi processing and executing factors.
[0601] According to one specific embodiment, the RNA molecule, such as a non-coding RNA molecule (i.e., rRNA, tRNA, lncRNA, snoRNA, etc.), is designed such that a sequence of the RNAi molecule is modified to be recognized by several cellular RNAi processing and executing factors.
[0602] It should be understood that additional mutations can be introduced through additional edit events (i.e., simultaneously or sequentially).
[0603] The DNA editing agent of the present invention can be introduced into several plant cells using several DNA delivery methods (e.g., via several expression vectors) or several DNA-free methods.
[0604] According to one embodiment, sgRNA (or any other DNA recognition module used, depending on the DNA editing system used) can be provided to the cell as RNA.
[0605] Therefore, it should be understood that the aforementioned techniques involve introducing the DNA editing agent using several transient DNA or DNA-free methods, such as RNA transfection (e.g., mRNA+sgRNA transfection) or ribonucleoprotein (RNP) transfection (e.g., protein-RNA complex transfection, such as Cas9 / sgRNA ribonucleoprotein (RNP) complex transfection).
[0606] For example, Cas9 can be introduced as a DNA expression plasmid, an in vitro transcript (i.e., RNA), or as a recombinant protein that binds to the RNA portion of a ribonucleoprotein particle (RNP). For example, sgRNA can be delivered as a DNA plasmid or an in vitro transcript (i.e., RNA).
[0607] According to this teaching, any method known in the art for RNA or RNP transfection may be used, such as, but not limited to, microinjection (as described by Cho et al., "Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins", Genetics, 2013, 195: 1177-1180, incorporated herein by reference), and electroporation (as described by Kim et al., "Highly efficient RNA-guided genomeediting in human cells via delivery of purified Cas9 ribonucleoproteins", Genome Res., 2014, 24:1012-1019, incorporated herein by reference, or lipid-mediated transfection, for example, using liposomes (as described by Zuris et al., “Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo,” Nat Biotechnol., 2014, doi: 10.1038 / nbt.3081, incorporated herein by reference). Several other methods of RNA transfection are described in U.S. Patent Application No. 20160289675, the entire contents of which are incorporated herein by reference.
[0608] One advantage of the multiple RNA transfection methods of the present invention is that RNA transfection is essentially transient and vector-free. An RNA transgene can be delivered to and expressed in a cell as a minimal expression cassette without requiring any other sequences (e.g., multiple viral sequences).
[0609] According to one embodiment, the DNA editing agent of the present invention is introduced into the plant cells using multiple expression vectors.
[0610] The “expression vector” (also referred to herein as a “nucleic acid construct”, “vector” or “construct”) described in some embodiments of the present invention comprises a plurality of additional sequences (e.g., a shuttle vector) that provide the vector to be adapted for replication in prokaryotes, eukaryotes or preferably both.
[0611] Multiple constructs useful in the various methods described in some embodiments of the present invention can be constructed using recombinant DNA techniques well known to those skilled in the art. The multiple nucleic acid sequences can be inserted into multiple vectors, which can be commercially available vectors suitable for transformation into multiple plants and for transient expression of the gene of interest in multiple transformed cells. The genetic construct can be an expression vector in which the nucleic acid sequence is operably linked to one or more regulatory sequences, thereby allowing expression in the multiple plant cells.
[0612] According to one embodiment, in order to express a functional DNA editing agent, when the cleaving module (nuclease) is not a component of the DNA recognition unit, the expression vector can be used to encode both the cleaving module and the DNA recognition unit (e.g., sgRNA in the case of CRISPR / Cas).
[0613] Alternatively, the cutting module (nuclease) and the DNA recognition unit (e.g., sgRNA) can be cloned into multiple separate expression vectors. In this case, at least two different expression vectors must be transformed into the same plant cell.
[0614] Alternatively, when a nuclease is not used (i.e., not applied to the cells from an exogenous source), the DNA recognition unit (e.g., sgRNA) can be cloned and expressed using a single expression vector.
[0615] Many typical expression vectors may also contain a transcription and translation initiation sequence, a transcription and translation terminator, and an optional polyadenylation signal.
[0616] According to one embodiment, the DNA editing agent includes a nucleic acid agent for encoding at least one DNA recognition unit (e.g., gRNA), which is operatively linked to a cis-acting regulatory element (e.g., a promoter) active in multiple plant cells.
[0617] According to one embodiment, the nuclease (e.g., endonuclease) and the DNA recognition unit (e.g., sgRNA) are encoded from the same expression vector. Such a vector may include a single cis-regulatory element (e.g., promoter) active in multiple plant cells for the expression of both the nuclease and the DNA recognition unit. Alternatively, the nuclease and the DNA recognition unit may each be operatively linked to a cis-regulatory element (e.g., promoter) active in multiple plant cells.
[0618] According to one embodiment, the nuclease (e.g., endonuclease) and the DNA recognition unit (e.g., sgRNA) are encoded from different expression vectors, wherein each expression vector is operatively linked to a cis-regulatory element (e.g., promoter) that is active in multiple plant cells.
[0619] As used herein, the phrase “plant-expressible” or “active in multiple plant cells” refers to a promoter sequence containing any other regulatory element added to or contained therein that is capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocot or dicot plant cell, tissue or organ.
[0620] The plant promoter used may be a set of established promoters, a tissue-specific promoter, an inducible promoter, a chimeric promoter, or a developmentally regulated promoter.
[0621] Examples of several preferred promoters useful for the various methods described in some embodiments of the present invention are shown in Tables I, II, III and IV.
[0622] Table I: Several exemplary constitutive promoters for implementing some embodiments of the present invention Table II: Several exemplary seed promoters for implementing some embodiments of the present invention Table III: Several exemplary flower-specific promoters for implementing the present invention Table IV: Several Alternative Rice Growth Promoters for Implementing the Invention The inducible promoter is a promoter induced in a specific plant tissue by a developmental stage or by a specific stimulus (e.g., multiple stress conditions, including, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions, or pathogenic conditions), and includes, but is not limited to, the light-inducible promoter from the pea rbcS gene, the promoter from the alfalfa rbcS gene, multiple promoters active in drought (DRE, MYC, and MYB); multiple promoters active in high salinity and osmotic stress (INT, INPS, prxEa, Ha hsp17.7G4, and RD21); and multiple promoters active under pathogenic stress (hsr203J and str246C).
[0623] According to one embodiment, the promoter is a pathogen-inducible promoter. These promoters direct the expression of multiple genes in multiple plants after infection with a pathogen (e.g., bacteria, fungi, viruses, nematodes, and insects). Such promoters comprise promoters from multiple pathogenesis-related proteins (PR proteins) that are induced upon infection with a pathogen; for example, multiple PR proteins, multiple SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al., 1983, Neth. J. Plant Pathol., 89: 245-254; Uknes et al., 1992, Plant Cell, 4: 645-656; and Van Loon, 1985, Plant Mol. Virol., 4: 111-116.
[0624] According to one embodiment, when more than one promoter is used in the expression vector, the plurality of promoters are identical (e.g., all identical, or at least two identical).
[0625] According to one embodiment, when more than one promoter is used in the expression vector, the plurality of promoters are different (e.g., at least two different, all different).
[0626] According to one embodiment, the promoter in the expression vector includes, but is not limited to, CaMV 35S, 2xCaMV 35S, CaMV 19S, ubiquitin, AtU626, or TaU6.
[0627] According to a specific embodiment, the promoter in the expression vector includes a 35S promoter.
[0628] According to a specific embodiment, the promoter in the expression vector includes a U6 promoter.
[0629] Multiple expression vectors may also include multiple transcription and translation initiation sequences, multiple transcription and translation termination sequences, and an optional polyadenylation signal.
[0630] According to a specific embodiment, the expression vector includes a termination sequence, such as, but not limited to, a G7 termination sequence, an AtuNos termination sequence, or a CaMV-35S termination sequence.
[0631] Multiple plant cells can be stably or transiently transformed using the multiple nucleic acid constructs described in some embodiments of the present invention. In stable transformation, the nucleic acid molecules described in some embodiments of the present invention are integrated into the plant genome, thus representing a stable and heritable trait. In transient transformation, the nucleic acid molecules are expressed by the transformed cells but are not integrated into the genome, thus representing a transient trait.
[0632] There are many methods for introducing multiple exogenous genes into monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol., 1991, 42: 205-225; Shimamoto et al., Nature, 1989, 338: 274-276).
[0633] The main methods for stably integrating exogenous DNA into plant genomic DNA include two main approaches: (i) Agrobacterium-mediated gene transfer: Klee et al., 1987, Annu. Rev. Plant Physiol., 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, Schell, J. and Vasil, LK (eds.), Academic Publishers, San Diego, CA, 1989, pp. 2-25; Gatenby in Plant Biotechnology, Kung, S. and Arntzen, CJ (eds.), Butterworth Publishers, Boston, MA, 1989, pp. 93-112.
[0634] (ii) Direct DNA uptake: Paszkowski et al., in *Cell Culture and Somatic Cell Genetics of Plants*, Vol. 6, *Molecular Biology of Plant Nuclear Genes*, edited by Schell, J. and Vasil, LK, Academic Publishers, San Diego, CA, 1989, pp. 52–68; including methods for direct DNA uptake into protoplasts, Toriyama, K. et al., 1988, *Bio / Technology*, 6: 1072–1074. DNA uptake induced by brief electric shock in plant cells: Zhang et al., *Plant Cell Rep.*, 1988, 7: 379–384. Fromm et al., *Nature*, 1986, 319: 791–793.DNA injection into plant cells or tissues via particle bombardment, Klein et al., Bio / Technology, 1988, 6:559-563; McCabe et al., Bio / Technology, 1988, 6:923-926; Sanford, Physiol. Plant, 1990, 79:206-209; Using a micropipette system: Neuhaus et al., Theor. Appl. Genet., 1987, 75:30-36; Neuhaus and Spangenberg, Physiol. Plant., 1990, 79:213-217; US Patent No. 5,464,765: Glassfibers or silicon carbide whisker transformation of cell cultures, embryos, or callus. (tissue), or by directly incubating DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, Chapman, GP, Mantell, SH, and Daniels, W. (eds.), Longman, London, 1985, pp. 197–209; and Ohta, Proc. Natl. Acad. Sci., USA, 1986, 83: 715–719.
[0635] The Agrobacterium system comprises the use of multiple plasmid vectors containing multiple defined DNA fragments integrated into the plant's genomic DNA. The various inoculation methods for the plant tissues vary depending on the plant species and the Agrobacterium delivery system. A widely used method is the leaf disc method, which can be performed using any tissue explant, providing a good source for initiating whole-plant differentiation. (Horsch et al., Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht, 1988, pp. 1-9). An auxiliary method is to combine the Agrobacterium delivery system with vacuum infiltration. The Agrobacterium system is particularly effective in creating transgenic dicotyledonous plants.
[0636] According to one embodiment, an Agrobacterium-free expression method is used to introduce multiple exogenous genes into multiple plant cells. According to one embodiment, the Agrobacterium-free expression method is transient. According to a specific embodiment, a bombardment method is used to introduce multiple exogenous genes into multiple plant cells. According to another specific embodiment, bombardment of a plant root is used to introduce multiple exogenous genes into multiple plant cells. Exemplary bombardment methods that can be used according to some embodiments of the present invention are discussed in the following examples sections.
[0637] Furthermore, according to the teachings of some embodiments of the present invention, various cloning kits or gene synthesis can be used.
[0638] According to one embodiment, the nucleic acid construct is a binary vector. Examples of binary vectors include pBIN19, pBI101, pBinAR, pGPTV, pCAMBIA, pBIB-HYG, pBecks, pGreen, or pPZP (Hajukiewicz, P. et al., Plant Mol. Biol., 25, 989, 1994; and Hellens et al., Trends in Plant Science, 5, 446, 2000).
[0639] Several examples of other vectors used in other DNA delivery methods (e.g., transfection, electroporation, bombardment, viral inoculation as described below) are: pGE-sgRNA (Zhang et al., Nat. Comms., 2016, 7:12697), pJIT163-Ubi-Cas9 (Wang et al., Nat. Biotechnol., 2004, 32, 947-951), pICH47742:2x3n5S-5'UTR-hCas9(STOP)-NOST (Belhan et al., Plant Methods, 2013, 11;9(1):39), pAHC25 (Christensen, AH and PHQuail, 1996, Ubiquitin promoter-based vectors for high-level expression of selectable and / or screenable marker genes in monocotyledons). genesinmonocotyledonous plants), Transgenic Research, 5:213 to 218), pHBT-sGFP(S65T)-NOS (Sheen et al., Protein phosphatase activity is required for light-induced gene expression in maize, EMBOJ., 12(9), 3497 to 3505, 1993).
[0640] According to one embodiment, the method of some embodiments of the present invention further includes introducing a plurality of donor oligonucleotides into the plant cells.
[0641] According to one embodiment, when the modification is an insertion, the method further includes introducing a plurality of donor oligonucleotides into the plant cell.
[0642] According to one embodiment, when the modification is a deletion, the method further includes introducing a plurality of donor oligonucleotides into the plant cells.
[0643] According to one embodiment, when the modification is a deletion and insertion (e.g., an exchange), the method further includes introducing a plurality of donor oligonucleotides into the plant cell.
[0644] According to one embodiment, when the modification is a point mutation, the method further includes introducing a plurality of donor oligonucleotides into the plant cells.
[0645] As used herein, the term "multiple donor oligonucleotides" or "multiple donor oligonucleotides" refers to multiple exogenous nucleotides, i.e., introduced from outside the plant cell to produce a precise change in the genome. According to one embodiment, the multiple donor oligonucleotides are synthetic.
[0646] According to one embodiment, the plurality of donor oligonucleotides are plurality of RNA oligonucleotides.
[0647] According to one embodiment, the plurality of donor oligonucleotides are plurality of DNA oligonucleotides.
[0648] According to one embodiment, the plurality of donor oligonucleotides are a plurality of synthetic oligonucleotides.
[0649] According to one embodiment, the plurality of donor oligonucleotides includes a plurality of single-stranded donor oligonucleotides (ssODNs).
[0650] According to one embodiment, the plurality of donor oligonucleotides includes a plurality of double-stranded donor oligonucleotides (dsODNs).
[0651] According to one embodiment, the plurality of donor oligonucleotides include a plurality of double-stranded DNA (dsDNA).
[0652] According to one embodiment, the plurality of donor oligonucleotides include a double-stranded DNA-RNA duplex.
[0653] According to one embodiment, the plurality of donor oligonucleotides include double-stranded DNA-RNA hybrids.
[0654] According to one embodiment, the plurality of donor oligonucleotides include single-stranded DNA-RNA hybrids.
[0655] According to one embodiment, the plurality of donor oligonucleotides include single-stranded DNA (ssDNA).
[0656] According to one embodiment, the plurality of donor oligonucleotides include double-stranded RNA (dsRNA).
[0657] According to one embodiment, the plurality of donor oligonucleotides include single-stranded RNA (ssRNA).
[0658] According to one embodiment, the plurality of donor oligonucleotides include the DNA or RNA sequences for exchange (as discussed above).
[0659] According to one embodiment, the plurality of donor oligonucleotides are in a non-expression vector form or in oligonucleotide form.
[0660] According to one embodiment, the plurality of donor oligonucleotides includes a DNA donor plasmid (e.g., a circular or linearized plasmid).
[0661] According to one embodiment, the plurality of donor oligonucleotides includes approximately 50 to 5000, approximately 100 to 5000, approximately 250 to 5000, approximately 500 to 5000, approximately 750 to 5000, approximately 1000 to 5000, approximately 1500 to 5000, approximately 2000 to 5000, approximately 2500 to 5000, approximately 3000 to 5000, approximately 4000 to 5000, and approximately 50 to 4000. 000, approximately 100 to 4000, approximately 250 to 4000, approximately 500 to 4000, approximately 750 to 4000, approximately 1000 to 4000, approximately 1500 to 4000, approximately 2000 to 4000, approximately 2500 to 4000, approximately 3000 to 4000, approximately 50 to 3000, approximately 100 to 3000, approximately 250 to 3000, approximately 500 to 3000 Approximately 750 to 3000, approximately 1000 to 3000, approximately 1500 to 3000, approximately 2000 to 3000, approximately 50 to 2000, approximately 100 to 2000, approximately 250 to 2000, approximately 500 to 2000, approximately 750 to 2000, approximately 1000 to 2000, approximately 1500 to 2000, approximately 50 to 1000, approximately 100 to 1000, approximately 25 0 to 1000, approximately 500 to 1000, approximately 750 to 1000, approximately 50 to 750, approximately 150 to 750, approximately 250 to 750, approximately 500 to 750, approximately 50 to 500, approximately 150 to 500, approximately 200 to 500, approximately 250 to 500, approximately 350 to 500, approximately 50 to 250, approximately 150 to 250, or approximately 200 to 250 nucleotides.
[0662] According to one specific embodiment, the plurality of donor oligonucleotides comprising the ssODN (e.g., ssDNA or ssRNA) comprises about 200 to 500 nucleotides.
[0663] According to one specific embodiment, the plurality of donor oligonucleotides comprising the dsODN (e.g., dsDNA or dsRNA) comprises about 250 to 5000 nucleotides.
[0664] According to one embodiment, for gene exchange of an endogenous RNA silencing molecule (e.g., miRNA) with a selected RNA silencing sequence (e.g., siRNA), the expression vector, ssODN (e.g., ssDNA or ssRNA), or dsODN (e.g., dsDNA or dsRNA) does not need to be expressed in a plant cell and can serve as a non-expression template. According to a specific embodiment, in this case, if provided in DNA form, only the DNA editing agent (e.g., multiple Cas9 / sgRNA modules) needs to be expressed.
[0665] According to some embodiments, for gene editing of an endogenous noncoding RNA molecule (e.g., an RNA silencing molecule) without the use of a nuclease, the DNA editing agent (e.g., sgRNA) can be introduced into the eukaryotic cell with or without, for example, oligonucleotide donor DNA or RNA (as described herein).
[0666] According to one embodiment, multiple donor oligonucleotides are introduced into the plant cells using any of the methods described above (e.g., using the expression vector or RNP transfection).
[0667] According to one embodiment, the sgRNA and the plurality of DNA donor oligonucleotides are co-introduced into the plant cells (e.g., by bombardment). It should be understood that any other factor (e.g., nuclease) can co-introduce the RNA. According to one embodiment, the sgRNA is introduced into the plant cell before the plurality of DNA donor oligonucleotides (e.g., within minutes or hours). It should be understood that any other factor (e.g., nuclease) may be introduced before, simultaneously with, or after the sgRNA or the plurality of DNA donor oligonucleotides.
[0668] According to one embodiment, the sgRNA is introduced into the plant cell after the plurality of DNA donor oligonucleotides (e.g., within minutes or hours). It should be understood that any other factor (e.g., nuclease) may be introduced before, simultaneously with, or after the sgRNA or the plurality of DNA donor oligonucleotides.
[0669] According to one embodiment, a composition is provided comprising at least one sgRNA for genome editing and a plurality of DNA donor oligonucleotides.
[0670] According to one embodiment, a composition is provided comprising at least one sgRNA for genome editing, a nuclease (e.g., a nuclease endonuclease), and a plurality of DNA donor oligonucleotides.
[0671] There are several methods for directly transferring DNA into multiple plant cells, and technicians know which method to choose. In electroporation, the multiple protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the multiple cells using a very small micropipette. In particle bombardment, the DNA is adsorbed onto multiple particles, such as multiple magnesium sulfate crystals or multiple gold or tungsten particles, and these particles physically accelerate their entry into multiple protoplasts, multiple cells, or multiple plant tissues.
[0672] Therefore, in various embodiments of the present invention, multiple nucleic acids can be delivered into a plant cell by any method known to those skilled in the art, including, but not limited to: transformation of multiple protoplasts (see, for example, U.S. Patent No. 5,508,184); DNA uptake mediated by desiccation / inhibition (see, for example, Potrykus et al., 1985, Mol. Gen. Genet., 199: 183-8); electroporation (see, for example, U.S. Patent No. 5,384,253); stirring with multiple silicon carbide fibers (see, for example, U.S. Patent Nos. 5,302,523 and 5,464,765); and transformation mediated by Agrobacterium (see, for example, U.S. Patent Nos. 5,508,184). Methods for delivering DNA, RNA, multiple peptides and / or multiple proteins or multiple combinations of multiple nucleic acids and multiple peptides into multiple plant cells by accelerating multiple DNA-coated particles (see, for example, U.S. Patent Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861 and 6,403,865), and by multiple nanoparticles, multiple nanocarriers and multiple cell-penetrating peptides (WO201126644A2; WO2009046384A1; WO2008148223A1).
[0673] Other transfection methods include the use of multiple transfection reagents (e.g., Lipofectin, ThermoFisher), multiple dendritic polymers (Kukowska-Latallo, JF et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 4897-1902), multiple cell-penetrating peptides (Mäe et al., 2005, Internalization of cell-penetrating peptides into tobacco protoplasts, Biochimica et Biophysica Acta, 1669(2): 101-7), or multiple polyamines (Zhang and Vinogradov, 2010, Short biodegradable polyamines for gene delivery and transfection of brain capillary endothelial cells, J Control). Release, 143(3): 359 to 366).
[0674] According to one specific embodiment, the method for introducing DNA into multiple plant cells (e.g., multiple protoplasts) includes polyethylene glycol (PEG)-mediated DNA uptake. For further details, see Karesch et al. (1991, Plant Cell Rep., 9:575-578); Mathur et al. (1995, Plant Cell Rep., 14:221-226); Negrutiu et al. (1987, Plant Cell Mol. Biol., 8:363-373). The multiple plant cells (e.g., multiple protoplasts) are then cultured under conditions that allow them to grow multiple cell walls, begin to divide to form a callus, develop multiple shoots and multiple roots, and regenerate the entire plant.
[0675] After stable transformation, plant propagation is carried out. The most common method of plant propagation is through seeds. However, a disadvantage of regeneration through seed propagation is the lack of uniformity in the crop due to heterozygosity, as multiple seeds result from multiple genetic variations in multiple plants governed by Mendelian rules. Essentially, each seed is genetically different and has its own specific traits. Therefore, it is preferable to produce transformed plants that possess the same traits and characteristics as the parent transgenic plant. Thus, it is preferable to regenerate the transformed plants through micropropagation, which provides a rapid and consistent propagation of genetically identical transformed plants.
[0676] Micropropagation is a process of growing multiple new generations of plants from a single excised tissue from a selected parent plant or variety. This process allows for the mass propagation of multiple plants with desired traits. The multiple newly produced plants are genetically identical to the original plant and possess all the characteristics of the original plant. Micropropagation (or cloning) can produce large quantities of high-quality plant material in a short time and provide rapid propagation of multiple selected varieties while retaining the multiple characteristics of the original transgenic or transformed plant. The advantages of multiple cloned plants are the speed of plant propagation and the quality and uniformity of the multiple plants produced.
[0677] Micropropagation is a multi-stage procedure that requires alteration of culture media or growth conditions between multiple stages. Therefore, the micropropagation process comprises four basic stages: Stage 1, initial tissue culture; Stage 2, tissue culture propagation; Stage 3, differentiation and plant formation; and Stage 4, greenhouse cultivation and hardening. During Stage 1 (initial tissue culture), the tissue culture is established and proven to be contamination-free. During Stage 2, the initial tissue culture is propagated until a sufficient number of tissue samples are produced to meet production targets. During Stage 3, the multiple tissue samples grown in Stage 2 are separated and grown into multiple individual plantlets. During Stage 4, the multiple transformed plantlets are transferred to a greenhouse for hardening, where the plants' light tolerance is gradually increased, allowing them to grow in a natural environment.
[0678] While stable transformation is currently preferred, some embodiments of the invention also envision transient transformation of multiple leaf cells, multiple meristematic cells, or the entire plant.
[0679] Transient transformation can be achieved through any of the above-mentioned direct DNA transfer methods or through viral infection using multiple modified plant viruses.
[0680] Several viruses, including CaMV, TMV, TRV, and BV, have been shown to be useful for transformation in multiple plant hosts. Multiple plant transformations using these plant viruses are described in U.S. Patent Nos. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Publication No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al. (Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172–189, 1988). Pseudovirus particles for expressing exogenous DNA in many hosts including plants are described in WO 87 / 06261.
[0681] Based on the above references and the work of Dawson, WO et al. (Virology, 1989, 172: 285-292; Takamatsu et al. (EMBO J., 1987, 6: 307-311); French et al. (Science, 1986, 231: 1294-1297); and Takamatsu et al. (FEBS Letters, 1990, 269: 73-76), it has been demonstrated that multiple plant RNA viruses with multiple non-viral exogenous nucleic acid sequences can be introduced and expressed in multiple plants.
[0682] When the virus is a DNA virus, the virus itself can be appropriately modified. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate the construction of the desired viral vector using the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can attach to the viral DNA, and replication can then occur through the bacteria. Transcription and translation of this DNA will produce a coat protein, which will encapsulate the viral DNA. If the virus is an RNA virus, it is typically cloned as a cDNA and inserted into a plasmid. The plasmid is then used to manufacture all the constructs. The RNA virus is then generated by transcribing the viral sequence of the plasmid and translating the plurality of viral genes to produce the coat protein(s) that encapsulate the viral RNA.
[0683] The above references and U.S. Patent No. 5,316,931 demonstrate the construction of multiple plant RNA viruses for introducing and expressing multiple non-viral exogenous nucleic acid sequences (e.g., sequences included in the constructs described in some embodiments of the present invention) in multiple plants.
[0684] In one embodiment, a plant virus nucleic acid is provided in which the natural coat protein coding sequence has been deleted from a viral nucleic acid, and a non-natural plant virus coat protein coding sequence and a non-natural promoter (preferably a subgenomic promoter of the non-natural viral coat protein coding sequence) have been inserted, which is capable of expressing, packaging, and ensuring systematic infection of the host via the recombinant plant virus nucleic acid in the plant host. Alternatively, the coat protein gene can be inactivated by inserting the non-natural nucleic acid sequence therein, thereby producing a protein. The recombinant plant virus nucleic acid may contain one or more other non-natural subgenomic promoters. Each non-natural subgenomic promoter is capable of transcribing or expressing multiple adjacent gene or nucleic acid sequences in the plant host, and these sequences cannot recombine with each other or with multiple natural subgenomic promoters. If more than one nucleic acid sequence is included, multiple non-natural (exogenous) nucleic acid sequences can be inserted near the natural plant virus subgenomic promoter or the natural plant virus subgenomic promoter and a non-natural plant virus subgenomic promoter. The multiple non-natural nucleic acid sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce multiple desired products.
[0685] In a second embodiment, a recombinant plant virus nucleic acid is provided as in the first embodiment, except that the natural coat protein coding sequence is placed near one of the plurality of non-natural coat protein subgenomic promoters instead of a non-natural coat protein coding sequence.
[0686] In a third embodiment, a recombinant plant virus nucleic acid is provided, wherein the natural coat protein gene is adjacent to its subgenomic promoter, and one or more non-natural subgenomic promoters have been inserted into the viral nucleic acid. The plurality of inserted non-natural subgenomic promoters are capable of transcribing or expressing multiple adjacent genes in a plant host and cannot recombine with each other or with the plurality of natural subgenomic promoters. Multiple non-natural nucleic acid sequences can be inserted near the plurality of non-natural subgenomic plant virus promoters, such that the plurality of sequences are transcribed or expressed in the host plant under the control of the plurality of subgenomic promoters to produce multiple desired products.
[0687] In a fourth embodiment, a recombinant plant virus nucleic acid is provided as in the third embodiment, except that the natural coat protein coding sequence is replaced by a non-natural coat protein coding sequence.
[0688] The multiple viral vectors encode the recombinant plant virus nucleic acid and are embedded in the multiple coat proteins to produce a recombinant plant virus. The recombinant plant virus nucleic acid or recombinant plant virus is used to infect multiple suitable host plants. The recombinant plant virus nucleic acid is capable of replicating in the host, systematically spreading in the host, and transcribing or expressing (multiple) exogenous genes (isolated nucleic acids) in the host to produce the desired protein.
[0689] In addition to the above, the nucleic acid molecules described in some embodiments of the present invention can also be introduced into a chloroplast genome, thereby enabling chloroplast expression.
[0690] Techniques for introducing multiple exogenous nucleic acid sequences into the genome of chlorophyll are known. This technique includes the following steps: First, multiple plant cells are chemically treated to reduce the number of chlorophyll molecules in each cell to approximately one. Then, the exogenous nucleic acid is introduced into the multiple cells by particle bombardment, with the aim of introducing at least one exogenous nucleic acid molecule into the multiple chlorophyll molecules. The exogenous nucleic acid is selected to integrate into the genome of the chlorophyll via homologous recombination, which is susceptible to the influence of multiple enzymes inherent to the chlorophyll. For this purpose, the exogenous nucleic acid, in addition to containing a gene of interest, contains at least one nucleic acid stretch derived from the genome of the chlorophyll. Additionally, the exogenous nucleic acid contains an optional marker that determines, through multiple sequential selection steps, that all or substantially all copies of the multiple chlorophyll genomes will contain the exogenous nucleic acid after such selection. Further details relating to this technique can be found in U.S. Patent Nos. 4,945,050 and 5,693,507, which are incorporated herein by reference. Therefore, a polypeptide can be generated and integrated into the inner membrane of chlorophyll through the protein expression system of the chlorophyll.
[0691] Regardless of the transformation / infection method used, this teaching further selects multiple transformed cells that include a genome editing event.
[0692] According to one specific embodiment, selection is made such that only multiple cells that include a successfully and accurately modified (e.g., exchange, insertion, deletion, point mutation) locus are selected. Therefore, multiple cells that include any event of a modification (e.g., insertion, deletion, point mutation) at an unintended locus are not selected.
[0693] According to one embodiment, multiple modified cells can be selected at the phenotypic level by detecting a molecular event, by detecting a fluorescent reporter, or by growing in the presence of a selector (e.g., an antibiotic).
[0694] According to one embodiment, multiple modified cells are selected by analyzing the biogenesis and occurrence of newly generated dsRNA molecules.
[0695] According to one embodiment, multiple modified cells are selected by analyzing the biogenesis and development of secondary small RNA molecules (produced through further processing of dsRNA).
[0696] According to one embodiment, multiple modified cells are selected by analyzing the biogenesis and development of newly edited RNA molecules (e.g., the presence of new miRNA versions, the presence of several newly edited siRNAs, piRNAs, tasiRNAs, etc.).
[0697] According to one embodiment, multiple modified cells are selected by analyzing the biogenesis and development of the newly edited plant RNA transcript (i.e., the modified plant gene).
[0698] According to one embodiment, multiple modified cells are selected by analyzing the modified RNA molecules (e.g., RNA silencing molecules) targeting a plant RNA or a pest RNA, or the silencing activity and / or specificity of the modified plant RNA (by verifying at least one phenotype encoding the target RNA in the plant or organism), the phenotype being, for example, plant leaf coloring, such as partial or complete loss of chlorophyll in leaves and other organs (bleaching), presence / absence of necrosis patterns, flower color, fruit traits (e.g., shelf life, firmness, and flavor), growth rate, plant size (e.g., dwarfing), crop yield, and tolerance to biological stress (e.g., disease resistance, nematode mortality, beetle oviposition rate, or other resistance phenotypes associated with bacteria, viruses, fungi, parasites, insects, weeds, and cultivated or native plants).
[0699] According to one embodiment, the silencing specificity of the RNA molecule, the plant RNA, the dsRNA, or the secondary small RNA processed therefrom is determined genotypically, for example, by the expression or absence of a gene.
[0700] According to one embodiment, the silencing specificity of the RNA molecule, the plant RNA, the dsRNA, or the secondary small RNA processed therefrom is determined phenotypically.
[0701] According to one embodiment, a phenotype of the plant is determined before a genotype.
[0702] According to one embodiment, a genotype of the plant is determined before a phenotype.
[0703] According to one embodiment, selection of multiple modified cells is performed by analyzing the silencing activity and / or specificity of the RNA molecule (e.g., an RNA silencing molecule), the plant RNA, the dsRNA, or the secondary small RNA processed therefrom against a plant RNA or a harmful RNA (by measuring the RNA level of the plant RNA or harmful RNA). This can be performed using any method known in the art, such as Northern Ink Dot assay, nuclease protection assay, in situ hybridization, or quantitative RT-PCR.
[0704] According to one embodiment, multiple modified cells are selected by analyzing multiple plant cells or multiple clones that include the DNA editing event, which is also referred to herein as a “mutation” or “edit”, depending on the type of editing sought, such as insertion, deletion, insertion-deletion (Indel), inversion, substitution, and combinations thereof.
[0705] Methods for detecting sequence alterations are well known in the art and include, but are not limited to, DNA and RNA sequencing (e.g., next-generation sequencing), electrophoresis, an enzyme-based mismatch detection assay, and a hybridization assay, such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, southern ink spot assay, northern ink spot assay, and dot blot analysis. Various methods for detecting single nucleotide polymorphisms (SNPs) can also be used, such as PCR-based T7 endonuclease, heteroduplex and Sanger sequencing, or restriction digestion after PCR to detect the presence or absence of (multiple) unique restriction sites.
[0706] Another method for verifying the existence of a DNA editing event (e.g., an insertion or deletion) includes a mismatch cleavage assay, which utilizes a structural selectase (e.g., an endonuclease) that recognizes and cleaves mismatched DNA.
[0707] According to one embodiment, multiple transformed cells are selected by flow cytometry (FACS) that exhibit fluorescence (via the emission of the fluorescent reporter). After FACS sorting, multiple populations of positively selected transformed plant cells displaying the fluorescent label are collected, and DNA editing events as described above can be tested using an aliquot.
[0708] In the case of using antibiotic selection markers, after transformation, multiple plant cell clones are cultured in the presence of selection (e.g., antibiotics) until they develop into multiple colonies, i.e., multiple clones and multiple micro-calli. As described above, a subset of cells from the callus is then analyzed (verified) in response to the DNA editing event.
[0709] Therefore, according to one embodiment of the invention, the method further includes verifying the complementarity of the RNA molecule (e.g., an RNA silencing molecule), the plant RNA, the dsRNA, or the secondary small RNA processed therefrom against the plant RNA or harmful organism RNA in the plurality of transformed cells.
[0710] As described above, the RNA molecule (e.g., RNA silencing molecule), the plant RNA, dsRNA (e.g., its sense or antisense strand), or the secondary small RNA processed therefrom, after modification, has at least about 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity against the sequence of the plant or harmful organism RNA.
[0711] The specific binding of the designed RNA molecule to a target RNA or a harmful organism can be determined by any method known in the art, for example by computational algorithms (e.g., BLAST), and verified by multiple methods, such as Northern Ink Dot assay, in situ hybridization, QuantiGene Plex assay, etc.
[0712] It should be understood that, for the DNA editing event described, multiple positive clones can be homozygous or heterozygous. In the case of a heterozygous cell (e.g., when diploid), the cell may include one copy of a modified gene and one copy of an unmodified gene. Those skilled in the art will select the clones for further culture / regeneration according to their intended use.
[0713] According to one embodiment, when a transient method is required, in the case of a missing DNA editing agent (i.e., the loss of multiple DNA sequences encoding the DNA editing agent), further analysis and selection are performed on multiple clones exhibiting the presence of the desired DNA editing event. This can be done, for example, by analyzing the loss of expression of the DNA editing agent (e.g., on the mRNA or protein), such as by fluorescence detection via GFP or q-PCR, or HPLC.
[0714] According to one embodiment, when a transient method is required, the plurality of cells can be analyzed to determine whether they lack a nucleic acid construct or a portion thereof as described herein, for example, a nucleic acid sequence encoding the DNA editing agent. This can be confirmed by fluorescence microscopy, q-PCR, FACS, or any other method (e.g., Southern Ink Dot assay, PCR, sequencing, HPLC).
[0715] According to one embodiment, the plant is hybridized as described below to obtain a plant lacking the DNA editing agent (e.g., endonuclease).
[0716] Multiple positive clones can be preserved (e.g., cryopreserved).
[0717] Alternatively, multiple plant cells (e.g., multiple protoplasts) can first grow into a group of plant cells that develop into a callus, and then regenerate into multiple complete plants by using multiple plant tissue culture methods to regenerate multiple shoots from the callus (callogenesis). The growth of multiple protoplasts into the callus and the regeneration of multiple shoots require a proper balance of multiple plant growth regulators in the tissue culture medium, which must be customized for each plant species.
[0718] Multiple protoplasts can also be used for plant breeding using a technique called protoplast fusion. This is achieved by inducing the fusion of multiple protoplasts from different species using an electric field or a polyethylene glycol solution. This technique can be used to generate somatic cell hybrids in tissue culture.
[0719] Methods for protoplast regeneration are well known in the art. Several factors influence protoplast isolation, culture, and regeneration, namely the genotype, the donor tissue and its pretreatment, the enzyme treatment used for protoplast isolation, the protoplast culture method, the culture, the culture medium, and the physical environment. For a comprehensive review, see Maheshwari et al. (1986, Differentiation of Protoplasts and of Transformed Plant Cells: 3-36, Springer-Verlag, Berlin).
[0720] If deemed appropriate by the technicians, further breeding and selection can be carried out on multiple regenerated plants.
[0721] Therefore, various embodiments of the present invention further relate to various plants, various plant cells, and various plant processing products, which include the dsRNA molecule capable of silencing a harmful organism gene generated according to the present teachings.
[0722] According to one aspect of the invention, a method of generating a pest-tolerant or resistant plant is provided, the method comprising generating a long dsRNA molecule capable of silencing a pest gene in a plant cell according to some embodiments of the invention.
[0723] According to one aspect of the present invention, a method for producing a pest-tolerant or pest-resistant plant is provided, the method comprising: (a) Breeding the plants described in some embodiments of the present invention; and (b) Select several progeny plants that express the long dsRNA molecule capable of inhibiting the harmful organism gene, and which do not contain the DNA editing agent. This results in the production of plants that are resistant to or tolerant of harmful organisms.
[0724] According to one aspect of the invention, a method for producing the plant or plant cells described in some embodiments of the invention is provided, comprising culturing the plant or plant cells under conditions that allow for reproduction.
[0725] According to one embodiment, breeding includes crossbreeding or self-pollination.
[0726] As used herein, the term "hybridization" refers to the fertilization of multiple female plants (or multiple gametes) by multiple male plants (or multiple gametes). The term "gamete" refers to a haploid reproductive cell (egg or sperm) produced in multiple plants through mitosis from a gametophyte, during which two gametes of opposite sexes fuse to form a diploid zygote. The term generally includes a pollen (containing the sperm cell) and an ovule (containing the egg). Thus, "hybridization" generally refers to the fertilization of multiple ovules from one individual with pollen from another individual, while "self-fertilization" refers to the fertilization of multiple ovules from one individual with pollen from the same individual. Hybridization is widely used in plant breeding and results in the mixing of genomic information between two plants, where the hybridization originates from a chromosome from the mother and a chromosome from the father. This will result in a new combination of multiple genetic traits.
[0727] As described above, the plants can be hybridized to obtain a plant that does not contain several unwanted factors, such as DNA editing agents (e.g., endonucleases).
[0728] According to some embodiments of the present invention, the plant is non-GMO.
[0729] According to some embodiments of the present invention, the plant is a transgenic plant.
[0730] According to one embodiment, the plant is non-genetically modified (non-GMO).
[0731] According to one embodiment, the plant is genetically modified (GMO).
[0732] According to one aspect of the present invention, a cell of the plant described in some embodiments of the present invention is provided.
[0733] According to one aspect of the present invention, a seed of the plant described in some embodiments of the present invention is provided.
[0734] According to one embodiment, compared with several plants not produced by this method (i.e., compared with several wild-type plants), the several plants produced by this method have at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% increased resistance or tolerance to pests.
[0735] According to the present invention, any method known in the art for assessing a plant’s tolerance or resistance to a pathogen can be used. Several exemplary methods include, but are not limited to, those described by Ramírez V1, García-Andrade J, and Vera P., where reduced MYB46 expression in Arabidopsis leads to enhanced resistance to Botrytiscinereal (Plant Signal Behav., June 2011; 6(6): 911-3, Epub: June 1, 2011); or those described by Gallego-Giraldo L. et al., where downregulation of HCT in alfalfa promotes activation of the plant’s defense response (NewPhytologist, 2011, 190: 627-639, doi: 10.1111 / j.1469-8137.2010.03621.x), both of which are incorporated herein by reference.
[0736] According to a further embodiment, a method is provided for generating a long dsRNA molecule in a plant cell, wherein the long dsRNA is capable of silencing a target gene of interest, the method comprising: (a) selecting a plant gene and a first nucleic acid sequence, the plant gene and the nucleic acid sequence showing a predetermined sequence homology with a nucleic acid sequence of the target gene of interest; and (b) modifying a second plant endogenous nucleic acid sequence encoding an RNA molecule to impart silencing specificity to the first plant gene, such that a plurality of small RNA molecules capable of recruiting RNA-dependent RNA polymerase (RdRp) to form base complementarity with a transcript of the first plant gene, the plurality of small RNA molecules being processed from the RNA molecule to generate the long dsRNA molecule capable of silencing the target gene of interest.
[0737] According to some embodiments, the first nucleic acid sequence does not encode a silenced RNA prior to using the above method. According to some embodiments, the long dsRNA is not naturally generated from the first nucleic acid sequence prior to using the above method. Without being bound by theory or mechanism, although the first nucleic acid sequence in the above method does not necessarily naturally generate long dsRNA (or any silenced RNA), modification of the second plant endogenous nucleic acid sequence produces an RNA molecule (e.g., miRNA). This RNA molecule acts as an amplifier and binds to RdRp to generate long dsRNA from an RNA transcript of the first nucleic acid sequence. Therefore, in practice, according to some embodiments, the above method can generate a long dsRNA from a gene that previously did not produce long dsRNA.
[0738] According to some embodiments, the target gene of interest is an endogenous gene of the plant cell. According to several other embodiments, the target gene of interest is an exogenous gene of the plant cell (e.g., a gene of a pest, such as an invertebrate pest).
[0739] According to some embodiments, the RNA molecule encoded by the second plant endogenous nucleic acid sequence is a miRNA.
[0740] According to some embodiments, the homology of the predetermined sequence with a nucleic acid sequence of the target gene of interest includes homology of at least two fragments, each fragment being at least 28 nt, and each fragment having at least 90% homology with the sequence of the target gene of interest.
[0741] According to some embodiments, modifying a nucleic acid sequence includes using a DNA editing agent, such as, but not limited to, a CRISPR-endonuclease (e.g., Cas9). According to some embodiments, the DNA editing agent includes a CRISPR-endonuclease and a guide RNA designed to cleave a nucleic acid sequence of interest (e.g., the sequence of the second plant endogenous nucleic acid). According to some embodiments, modifying a nucleic acid sequence of interest includes using a DNA editing agent (possibly having a guide RNA for cleaving the nucleic acid of interest) and further introducing an additional nucleic acid sequence into the plant cell, the additional nucleic acid sequence being similar to the nucleic acid sequence to be modified, but the nucleic acid sequence to be modified including several desired nucleotide changes. Unwilling to be bound by theory or mechanism, the DNA editing agent cleaves the nucleic acid sequence of interest and introduces the partial additional nucleic acid sequence (including several desired nucleotide changes) into the nucleic acid sequence of interest via homology-dependent recombination (HDR).
[0742] As used in this article, the term "about" means ±10%.
[0743] The terms “comprises”, “comprising”, “includes”, “including”, “having”, and their inflections mean “including but not limited to”.
[0744] The term "consisting of" means "included in and limited to".
[0745] The term "consisting essentially of" means that a composition, method, or structure may contain other components, steps, and / or parts, provided that the other components, steps, and / or parts do not substantially alter the fundamental and novel features of the claimed composition, method, or structure.
[0746] As used herein, the singular forms “a,” “an,” and “the” include plural references unless otherwise expressly stated herein. For example, the terms “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
[0747] Throughout this application, different embodiments of the invention may be presented in a range format. It should be understood that the range format description is for convenience and brevity only and should not be construed as an immutable limitation on the scope of the invention. Therefore, the range description should be considered as specifically disclosing all possible sub-ranges and their respective numerical values. For example, a range such as 1 to 6 should be considered as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and their respective numbers, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the range width.
[0748] Whenever a range of values is specified herein, it means that any of the stated values (fractions or integers) within the specified range is included. The phrases “range between the first and second digits” and “range from the first digit to the second digit” are used interchangeably herein to mean that the first and second digits and all fractions and integers in between are included.
[0749] As used herein, the term “method” means the manner, means, techniques and procedures used to accomplish a given task, including but not limited to manner, means, techniques and procedures known or readily developed by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine from known means, techniques and procedures.
[0750] As used in this article, the term "treatment" includes eliminating, substantially inhibiting, slowing or reversing the progression of the disease, substantially improving the clinical and aesthetic symptoms of the disease, or substantially preventing the occurrence of the clinical or aesthetic symptoms of the disease.
[0751] It should be understood that, for clarity, certain features of the invention described in the context of various embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, different features of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination or applicable to any other described embodiments of the invention. Certain features described in different embodiments are not considered essential features of the embodiments unless the embodiment is invalid without the described element.
[0752] The following examples provide experimental support for different embodiments and aspects of the invention as described above and partially claimed in the appended claims.
[0753] It should be understood that any sequence identification number (SEQ ID NO) disclosed in this application may refer to a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is mentioned, even if the SEQ ID NO is expressed only in a DNA sequence format or an RNA sequence format. For example, SEQ ID NO: 1 is expressed in a DNA sequence format (e.g., T for thymine), but it may refer to a DNA sequence corresponding to a nucleic acid sequence or the RNA sequence of an RNA molecule. Similarly, although some sequences are expressed in an RNA sequence format (e.g., U for uracil), depending on the actual type of the molecule, it may refer to a sequence of an RNA molecule comprising a dsRNA or a sequence of a DNA molecule corresponding to the RNA sequence shown. In any case, DNA and RNA molecules having sequences disclosed along with any substituents are conceivable.
[0754] example The invention will now be illustrated in a non-limiting manner with reference to the following examples, together with the description above.
[0755] Generally, the nomenclature used herein and the laboratory procedures utilized in this invention encompass molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully described in the literature. See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., 1989; "Current Protocols in Molecular Biology," Volumes I-III, Ausubel, RM (ed.), 1994; Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland, 1989; Perbal, "A Practical Guide to Molecular Cloning," John Wiley and Sons, New York, 1988; Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (ed.), "Genome Analysis: A Laboratory Manual Series," Volumes I-IV, Cold Spring Harbor Laboratory Press. Press), New York, 1998; such as U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III, Cellis, JE (ed.), 1994; "Culture of Animal Cells: A Manual of Basic Technique", Freshney, Wiley-Liss, New York, 1994, 3rd edition; "Current Protocols in Immunology", Volumes I-III, Coligan, JE (ed.), 1994; Stites et al. (eds.), "Basic and Clinical Immunology" (8th edition), Appleton and Lange, Norwalk, CT, 1994; Mishell and Shiigi (eds.), "Selected Methods in Cellular Immunology", WH Freeman and Co.The method shown in the New York, 1980; readily available immunoassays are extensively described in patents and scientific literature, see, for example, U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521, “Oligonucleotide Synthesis,” Gait, MJ ed., 1984; “Nucleic Acid Hybridization,” Hames, BD and Higgins. SJ, ed., 1985; "Transcription and Translation", Hames, BD and Higgins, SJ, ed., 1984; "Animal Cell Culture", Freshney, RI, ed., 1986; "Immobilized Cells and Enzymes", IRL Press, 1986; "A Practical Guide to Molecular Cloning", Perbal, B., 1984 and "Methods in Enzymology", Volumes 1–317, Academic Press; "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, UC, 1990; Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", CSHL Press, 1996; all of these are incorporated by reference as if fully expounded herein. Other general references are provided throughout the document. The procedures described herein are considered to be well-known in the art and are provided for the convenience of the reader. All information contained herein is incorporated herein by reference.
[0756] General materials and experimental procedures Calculate pipelines to generate GEiGS templates The computational GEiGS pipeline utilizes biological metadata and can automatically generate multiple GEiGS DNA templates. These templates are used to minimally edit multiple non-coding RNA genes (e.g., multiple miRNA genes) to gain a new function: redirecting their silencing ability to a target sequence of interest.
[0757] like Figure 6 As shown, the pipeline begins with inputting and submitting input: (a) silencing the target sequence via GEiGS; (b) gene editing of the host organism and expression of the GEiGS; and (c) the option to either universally express the GEiGS. If specific GEiGS expression is required, several options can be selected (expression specific to a particular tissue, developmental stage, stress, heat / cold shock, etc.).
[0758] Once all required inputs are submitted, the computation process begins by searching and filtering multiple relevant miRNA datasets (e.g., small RNA sequencing, microarrays, etc.) that meet only the input criteria. Next, the selected mature miRNA sequences are aligned with the target sequence, and miRNAs with the highest complementarity levels are selected. These naturally occurring target-complementary mature miRNA sequences are then modified to perfectly match the target sequence. The modified mature miRNA sequences are then run through an algorithm predicting siRNA efficacy, and the top 20 with the highest silencing scores are selected. These finally modified miRNA genes are then used to generate 200–500 nt ssDNA or 250–5000 nt dsDNA sequences, as shown below.
[0759] A 200-500 nt ssDNA oligonucleotide and a 250-5000 nt dsDNA fragment are designed based on the genomic DNA sequences flanking the modified miRNA. The pre-miRNA sequence is located at the center of the oligonucleotide. The guide strand (silencing) sequence of the modified miRNA is 100% complementary to the target. However, the sequence of the modified passenger miRNA strand is further modified to preserve the original (unmodified) miRNA structure, maintaining the same base-pairing profile.
[0760] Next, multiple differentially expressed sgRNAs were designed to specifically target the original, unmodified miRNA gene, rather than the modified exchange form. Finally, restriction enzyme site analysis was performed between the modified miRNA gene and the original miRNA gene, and multiple differentially expressed restriction sites were identified.
[0761] Therefore, the pipeline output includes: (a) A 200 to 500 nt ssDNA oligonucleotide or a 250 to 5000 nt dsDNA fragment sequence of a miRNA with minimal modification.
[0762] (b) Two to three differentially expressed sgRNAs that specifically target the original miRNA gene rather than the modified miRNA gene.
[0763] (c) A list of multiple differential restriction enzyme sites between the modified miRNA gene and the original miRNA gene.
[0764] dsRNA design via GEiGS Model 1 (the numbers correspond to the numbers in Figure 1): 1. The harmful organism gene "X" is the target gene (when silenced, the harmful organism is controlled). 2. Identify a host-related gene X by homology search with the harmful organism gene "X" (plant gene "X"). According to some embodiments, if the plant gene X comprises at least two stretches of at least 28 nt, each stretche having at least 90% homology with the sequence of the harmful organism gene X, then the plant gene X is identified according to Model 1.
[0765] 3. Perform GEiGS in several plant cells to specifically redirect the silencing of a small RNA molecule (e.g., 22 nt miRNA) to the host-associated gene-X, thereby making the small RNA molecule an amplifier of RdRp-mediated transcription for the transcript of the plant gene "X".
[0766] 4. The amplified small RNA, which is retargeted for silencing specificity using GEiGS (also referred to herein as “small GEiGS RNA”), forms a RISC complex associated with RdRp (amplification enzyme).
[0767] 5. The RdRp synthesizes a complementary antisense RNA strand for the transcript of the plant gene "X", forming a long dsRNA.
[0768] 6. Then, the long dsRNA is at least partially processed into several secondary sRNAs by (several) dicers or other nucleases within the several plant cells. Among these secondary sRNAs, the silencing specificity of some of the secondary sRNAs is targeted at the harmful organism gene X.
[0769] 7. The dsRNA is also absorbed at least partially by several harmful organisms, and may be processed into several sRNAs in the harmful organisms, as described above.
[0770] 8. In addition to, for example, several long dsRNAs produced, several secondary sRNAs that may originate from the plant cells are also absorbed by several harmful organisms and silence the target gene "X".
[0771] Model 2 (the numbers correspond to the numbers in Figure 2): 1. The harmful organism gene "X" is the target gene (when silenced, the harmful organism is controlled). 2. GEiGS is performed in several plant cells to specifically redirect the silencing of a naturally occurring RNAi precursor to the harmful gene “X” (e.g., the TAS gene; which is amplified into a long dsRNA and processed into several tasiRNAs of its wild type), the naturally occurring RNAi precursor being known to amplify in its wild-type form (i.e., it produces long dsRNA). The transcript is labeled “Amplified GEiGS precursor” in Figure 2. According to some embodiments, an RNAi precursor that can be used with Model 2 is an RNAi precursor that forms a long dsRNA and is processed into several secondary small RNAs, such as, but not limited to, a precursor processed into a trans-acting siRNA (tasiRNA) or a certain phase small interfering RNA (phasiRNA). Genome editing-induced gene silencing (GEiGS) is performed on the gene encoding the RNAi precursor by using a DNA "GEiGS oligonucleotide" to introduce several nucleotides into the gene through the induction of a double-strand break in the gene using a nuclease (e.g., CAS9) and to provide the changes required for specific redirection using homology-dependent recombination (HDR). Therefore, depending on the "GEiGS oligonucleotide" used, the specificity of a portion of the RNAi precursor (e.g., tTAS) will be altered to target the harmful gene X. The redirected RNAi precursor will be processed by the cell Dicer into several secondary small RNAs (e.g., several tasiRNAs), which will also match the harmful gene X. In the example shown in Figure 2, only one of the several tasiRNAs will be altered, resulting in the processing of a TAS into the several wild-type and altered tasiRNAs.
[0772] 3. A wild-type amplified small RNA forms a RISC complex associated with RdRp (the amplification enzyme).
[0773] 4. The RdRp synthesizes a complementary antisense RNA strand for the transcript of the amplified GEiGS precursor, forming a long dsRNA.
[0774] 5. The amplified GEiGS dsRNA is at least partially processed into several secondary sRNAs by (several) dicers or other nucleases in plant cells. Among these secondary sRNAs, the silencing specificity of the secondary small RNA corresponding to the location of GEiGS is against the harmful organism gene X.
[0775] 6. At least a portion of the unprocessed GEiG long dsRNAs are absorbed by several harmful organisms and may be processed into several small RNAs in the harmful organisms, as described above.
[0776] 7. Several secondary sRNAs (e.g., several tasiRNAs in several TAS precursors) that may have been produced in the plant cells may also be absorbed by the harmful organism and silence the target gene "X".
[0777] Tables 1A and 1B below provide several exemplary pest genes that can be targeted by several methods of this invention (particularly Model 1). Table 2 below provides several exemplary pest genes that can be targeted by several methods of this invention (particularly Model 2). Table 2 also provides several RNAi precursors, such as several TAS RNA precursors, that are suggested to be targeted by GEiGS (denoted as "backbone"). Table 2 provides several suggested small interfering RNAs (denoted as "desired siRNAs") that can be introduced into the suggested backbone using GEiGS, thereby enabling the backbone to be processed into these siRNAs in the several pests, thereby influencing the silencing of the several target genes.
[0778] Table 1A: List of several potential pest target genes and their accession numbers (including several plant homologous genes, according to Model 1) Table 1B: List of several potential target genes of harmful organisms and their accession numbers Table 2: Several potential harmful target genes and several examples of tasiRNA-based silencing using GEiGS (2 models per example). Bombardment and plant regeneration of Arabidopsis thaliana and tomato Arabidopsis root preparation Multiple Arabidopsis thaliana (cv. Col-0) seeds, sterilized with chlorine, were sown on MS minus sucrose plates and vernalized for 3 days in the dark at 4°C, followed by vertical germination under constant light at 25°C. Two weeks later, the roots were cut into 1cm segments and placed on callus induction media (CIM: 1 / 2 MS (containing vitamin B5), 2% glucose, pH 5.7, 0.8% agar, 2 mg / L IAA, 0.5 mg / L 2,4-D, 0.05 mg / L kinetin) plates. After culturing in the dark at 25°C for 6 days, the root segments were transferred to multiple filter paper discs and placed on multiple CIMM plates (1 / 2 MS (vitamin-free), 2% glucose, 0.4M mannitol, pH 5.7 and 0.8% agar) for 4 to 6 hours in preparation for bombardment.
[0779] Tomato explant preparation: Several tomato seeds were surface-sterilized with a commercial bleach for 20 minutes, and then washed three times with sterile water under aseptic conditions. The seeds were then cultured on germination medium (MS + vitamins, 0.6% agarose, pH=5.8) at 25°C with a 16 / 8 hour light / dark cycle.
[0780] Several cotyledons from 8-day-old tomato plants were cut into sections approximately 1 cm² and placed on pre-bombardment culture (MS + vitamins, 3% sucrose, 0.6% agarose, pH 5.8, 1 mg / L BAP, 0.2 mg / L IAA) and incubated in the dark at 25 °C for 2 days. Then, several explants were transferred to the center of a target plate (containing MS + vitamins, 3% mannitol, 0.6% agarose, pH 5.8) for 4 hours.
[0781] bombardment Multiple plasmid constructs were introduced into the root tissue via PDS-1000 / He particle delivery (Bio-Rad; PDS-1000 / He system #1652257), a procedure that requires several preparation steps outlined below.
[0782] Gold reserve preparation 40 mg of 0.6 μm gold (Bio-Rad; Cat: 1652262) was mixed with 1 ml of 100% ethanol, pulsed centrifuged to form clumps, and the ethanol was removed. This washing procedure was repeated two more times.
[0783] After washing, the granules were resuspended in 1 ml of sterile distilled water and distributed into 1.5 ml test tubes (50 μl aliquots of the working sample volume).
[0784] magnetic bead preparation In short, do the following: Generally, a single test tube has enough gold to bombard two discs of Arabidopsis roots (two shots per disc), therefore, each test tube is allocated among four (1,100 psi) biolistic rupture disks (Bio-Rad; Cat: 1652329).
[0785] Bombardment requires multiple plates of the same sample. To maintain sample consistency and minimize overall preparation work, the tubes are combined and the volumes of the DNA and CaCl2 / spermidine mixture are adjusted accordingly.
[0786] The following procedure summarizes the process of preparing a test tube of gold. The procedure should be adjusted according to the amount of gold used in the test tubes.
[0787] All subsequent processes were carried out in an Eppendorf thermomixer at 4°C.
[0788] Prepare multiple plasmid DNA samples, each tube containing 11 μg of DNA at a concentration of 1000 ng / μl.
[0789] (1) Add 493 μl ddH2O to one part (7 μl) of spermidine (Sigma-Aldrich; SO266) to a final concentration of 0.1 M spermidine. Add 1250 μl 2.5 M CaCl2 to the spermidine mixture, vortex and place on ice.
[0790] (2) Place a test tube of gold prepared in advance in the hot mixer and rotate it at a speed of 1400 rpm.
[0791] (3) Add 11 μl of DNA to the test tube, vortex and return to the rotating hot mixer.
[0792] (4) To bind DNA / gold particles, add 70 μl of spermidine CaCl2 mixture to each test tube (in the hot mixer).
[0793] (5) Vigorously vortex the multiple test tubes for 15 to 30 seconds and place them on ice for about 70 to 80 seconds.
[0794] (6) Centrifuge the mixture at 7000 rpm for 1 minute, remove the supernatant and place it on ice.
[0795] (7) Add 500 μl of 100% ethanol to each test tube and resuspend the pellets by pipetting and vortexing.
[0796] (8) Centrifuge the multiple test tubes at 7000 rpm for 1 minute.
[0797] (9) Remove the supernatant, resuspend the pellets in 50 μl of 100% ethanol, and store on ice.
[0798] Macrocarrier preparation The following operations are performed in a laminar flow cabinet: (1) Sterilize and dry multiple macrocarriers (Bio-Rad; 1652335), multiple stopping screens (Bio-Rad; 1652336), and multiple macrocarrier disk racks.
[0799] (2) Place multiple macrocarriers flat into the multiple macrocarrier disk racks.
[0800] (3) Vortex the multiple DNA-coated gold mixtures and disperse (5 μl) onto the center of each gene gun rupture disc.
[0801] Ethanol evaporation is permitted.
[0802] PDS-1000 (Helium Particle Delivery System) In short, do the following: Adjust the regulating valve of the helium cylinder to an incoming pressure of at least 1300 psi. Create a vacuum by pressing the vac / vent / hold switch and holding the fire switch for 3 seconds. This ensures that helium is released into the piping system.
[0803] Multiple 1100psi rupture discs were placed in isopropanol and mixed to remove static electricity.
[0804] (1) Place one broken disk into the disk retaining cap.
[0805] (2) Construct a microcarrier launch assembly (with a stop screen and a gold-containing microcarrier).
[0806] (3) Place the covered petri dish of Arabidopsis root callus 6 cm below the emission assembly.
[0807] (4) Set the vacuum pressure to 27 inches of mercury and open the helium valve (approximately 1100 psi).
[0808] (5) Release the vacuum; remove the microcarrier emission assembly and the rupture disc fixing cover.
[0809] (6) Bombard the same tissue (i.e., bombard each plate twice).
[0810] (7) Then place multiple bombarded roots on multiple CIM plates in the dark at 25°C and leave for 24 hours.
[0811] Co-bombardment When bombarding multiple combinations of GEiGS plasmids, mix 5 μg (1000 ng / μl) of the sgRNA plasmid with 8.5 μg (1000 ng / μl) of the exchange plasmid, and add 11 μl of this mixture to the sample. If bombarding more GEiGS plasmids simultaneously, the concentration ratio of the multiple sgRNA plasmids to the multiple exchange plasmids used is 1:1.7, and add 11 μg (1000 ng / μl) of this mixture to the sample. If bombarding with multiple plasmids unrelated to GEiGS exchange, mix them in equal proportions, and add 11 μg (1000 ng / μl) of the mixture to each sample.
[0812] Transfection of several Col-0 protoplasts Several Arabidopsis (Col-0) protoplasts were transfected with several vectors encoding Crispr / Cas9 and a donor template to achieve several HDR-mediated swaps. The experiment was designed to swap several sequences in the Tas1b (AtTAS1b_AT1G50055) or Tas3a (AtTAS3a_AT3G17185) genes to generate several sRNAs targeting several 30 bp sequences in the aforementioned nematode target genes. The fundamental principle for generating a long dsRNA targeting several 30 bp sequences in the nematode, without being bound by theory or mechanism, is to ensure that when the dsRNA is processed into several secondary silent RNAs in the nematode, several functional silent RNAs are generated even if the length of the secondary silent RNAs formed in several nematodes differs from the length of the secondary silent RNAs formed in the plant.
[0813] Two exchanges were designed in the TAS1b locus and two exchanges were designed in the TAS3a locus. These exchanges were independent of each other. The DONOR template (1 kb) was synthesized in several plasmids (by Twist, USA).
[0814] The protoplast concentration was determined using a hematology counter and Trypan Blue (approximately 30 μl protoplasts, 65 μl mmol, and 5 μl trypan blue). The protoplasts were several diluted or concentrated protoplasts directed to a final density of 2 x 10⁶ cells / ml.
[0815] For PEG transfection, the molar ratio of sgRNA Vector (CRISPR / Cas, sgRNA, mCHERRY): DONOR Vector is 1:20, equivalent to 3.9 μg sgRNA Vector and approximately 21.61 μg DONOR Vector per transfection. Slowly add 1 ml of PEG solution to several 1 ml protoplasts. Prepare a fresh PEG solution (2 g PEG 4000 (Sigma), 0.2 M mannitol, and 0.5 ml 1 M CaCl2 per 5 ml). Incubate several tubes at room temperature in the dark for 20 minutes, then add 4 ml of W5 solution and mix the tubes by inverting. Resuspend the protoplast pellet in 5 ml of PCA (protoplast regeneration medium) to allow cell division, which is beneficial for HDR.
[0816] Cell analysis 24 to 72 hours after plasmid delivery, several cells were collected and resuspended in D-PBS medium. Half of the solution was used for luciferase activity analysis, and the other half for small RNA sequencing. Dual luciferase assay was performed using the Dual-Glo® Luciferase Assay System (Promega, USA) according to the manufacturer's instructions. Total RNA was extracted using a total RNA purification kit (Norgene Biotek Corp., Canada) according to the manufacturer's instructions. Small RNA sequencing was performed to identify the desired mature small RNAs in these samples.
[0817] Arabidopsis plant regeneration For shoot regeneration, a modified protocol from Valvekens et al. was used (Valvekens, D. et al., Proc Natl Acad Sci, USA, 1988, 85(15): 5536-5540). Multiple bombarded roots were placed on multiple shoot induction media (SIM) plates containing 1 / 2 MS (containing vitamin B5), 2% glucose, pH 5.7, 0.8% agar, 5 mg / L 2 iP, and 0.15 mg / L IAA. The plates were cycled between 16 hours of light at 25°C and 8 hours of darkness at 23°C. After 10 days, the plates were transferred to multiple MS plates (containing 3% sucrose and 0.8% agar) for one week, and then to multiple fresh, similar plates. Once multiple plants had regenerated, they were removed from the multiple roots and placed on multiple MS plates (containing 3% sucrose and 0.8% agar) until analysis.
[0818] Post-bombardment culture and plant regeneration of tomatoes Several bombarded explants were placed in the dark at 25 °C on MS medium (MS + vitamins, 3% sucrose, 0.4% agarose gel, pH 5.8, 1 mg / L BAP, 0.2 mg / L IAA) for two days. Several explants were then transferred to 16 / 8 light / dark cycles and subcultured every 2 weeks. Several regenerated shoots were transferred to root induction medium (MS + vitamins, 3% sucrose, 2.25% gelrite, pH 5.8, 2 mg / L IBA).
[0819] Wash several rooted plants with water to remove all agar residue, and place them in and cover them with soil. After a week of acclimatization, gradually remove the caps to allow the plants to harden.
[0820] Genotyping Following the manufacturer's recommendations, several tissue samples were treated and several amplicones were amplified using the PhirePlant Direct PCR Kit (Thermo Scientific). Several oligonucleotides used for these amplifications were designed to amplify the genomic region from a region within the modified sequence of the GEiGS system to a region outside the region used as the HDR template, to distinguish DNA incorporation. Several different modifications at the modified loci were confirmed by several different restriction enzyme digestion patterns of the amplicones (given by several specifically selected restriction endonucleases).
[0821] Genomic PCR reaction Following the manufacturer's instructions, genomic DNA from several cell samples (A, B, C, D, and E, as discussed in Example 3 below) were processed using an RNA / DNA purification kit (Norgen). The samples were quantified using a Qubit quantifier, and the DNA was stored at -20°C.
[0822] A non-specific primer flanking the exchange region was used for the several Tas1b (AtTAS1b_AT1G50055) and Tas3a (AtTAS3a_AT3G17185) sequences. As a negative control, several identical exchange-specific reactions were performed using wild-type (WT) DNA as a template. As a positive PCR control, all samples underwent a single-specific PCR against WT DNA. Q5® High-Fidelity 2X Master Mix was used for several PCR amplifications.
[0823] Each PCR reaction was run at 5 µl on a 0.8% agarose gel. Several band sizes were estimated by comparison with molecular weight markers (MW): 1 kb Plus DNA Ladder (NEB).
[0824] To confirm the multiple exchanges, a nested PCR reaction was performed. The first genomic PCR included several non-specific forward and reverse primers located flanking the HDR region. Several PCR products were diluted 1 / 100 with miL-Q ultrapure water, and then the aforementioned specific exchange PCR was performed. In the nested approach, the multiple non-specific primers used for the first PCR have multiple annealing sites flanking the multiple annealing sites of the nested primers.
[0825] Several primers used: Non-specific primers for Tas1b: - Tas1b_WT_Chimeric Nonspecific_DNA_R: 5´-accaatttgacccaaaaaggc-3´ (SEQ IDNO: 63) Tas1b exchange-specific primers: - Tas1b_splicing30_chimeric_DNA_F: 5´-GCAGCAGATCAATGAAATTCAACG-3´ (SEQ IDNO: 64) - Tas1b_Y2530_chimeric_DNA_F: 5´-agCCGCCTCTGTGGATTCTTG-3´ (SEQ ID NO:65) Non-specific primers for Tas3a: - Tas3a_WT_Chimeric Nonspecific_DNA_R: 5´-aaactcctcgccctcttggtg-3´ (SEQ IDNO: 66) Exchange-specific primers for Tas3a: - Tas3a_Ribo3a30_Chimeric_DNA_F: 5´-TCTTCAGCACCTTCACCTTACG-3´ (SEQ IDNO: 67) - Tas3a_Spliceo30_chimeric_DNA_F: 5´-TCCTTTTTGACCAACATTTGTTTGT-3´ (SEQ ID NO: 68) Positive control reaction WT Tas1b specificity: - Tas1b_WT_Chimeric Nonspecific_DNA_R: 5´-accaatttgacccaaaaaggc-3´ (SEQ IDNO: 69) - Tas1b_WT_Chimeric_DNA_F: 5´-tggacttagaatatgctatgttggac-3´ (SEQ ID NO:70) WT Tas3a specificity: - Tas3a_WT_Chimeric Nonspecific_DNA_R 5´-aaactcctcgcctcttggtg-3´ (SEQ IDNO: 71) - Tas3a_WT_chimeric_DNA_F 5´-tctatctctacctctaattcgttcgag-3´ (SEQ ID NO:72) DNA and RNA separation Multiple samples were collected in liquid nitrogen and stored at -80°C until processing. Tissue was ground in multiple tubes placed on dry ice using a tissue grinder pestle (Axygen, USA). DNA and total RNA were isolated from the ground tissue using an RNA / DNA purification kit (cat. 48700; Norgen Biotek Corp., Canada) according to the manufacturer's instructions. With the RNA fraction at a low 260 / 230 ratio (<1.6), the isolated RNA was precipitated overnight at -20°C with 1 μl of glycogen (cat. 10814010; Invitrogen, USA), 10% v / v sodium acetate, 3M pH 5.5 (cat. AM9740; Invitrogen, USA), and 3 volumes of ethanol. The solution was centrifuged at maximum speed for 30 minutes at 4°C. It was then washed twice with 70% ethanol, air-dried for 15 minutes, and resuspended in nuclease-free water (cat. 10977035; Invitrogen, USA).
[0826] RNA extraction Following the manufacturer's instructions, several cell samples (A, B, C, D, E, as described in Example 3 below) were treated with an RNA / DNA purification kit (Norgen) for RNA purification. The samples were quantified using qubits. RNA was stored at -80°C.
[0827] DNA enzyme treatment of RNA samples RT-PCR and subsequent PCR were used to specifically identify several small dsRNA fragments including the exchange (<200 bp) to demonstrate the biogeneration of dsRNAs capable of targeting several nematode target genes. For this purpose, the Turbo DNA-Free Kit (Invitrogen) was used according to the manufacturer's instructions. DNAse treatment was further performed to normalize sample concentrations.
[0828] Reverse transcription (RT) and quantitative real-time PCR (qRT-PCR) According to the manufacturer's manual (AMPD1; Sigma-Aldrich, USA), 1 μg of isolated total RNA was treated with DNase I. The sample was reverse transcribed according to the instructions of the High-Capacity cDNA Reverse Transcription Kit (cat. 4368814; Applied Biosystems, USA).
[0829] For gene expression, quantitative real-time PCR (qRT-PCR) analysis was performed on the CFX96 Touch™ Real-Time PCR Detection System (BioRad, USA) and the Green JumpStart™ Taq ReadyMix™ (S4438, Sigma-Aldrich, USA) according to the manufacturer's protocol, and the analysis was performed using the Bio-Rad CFX manager program (version 3.1).
[0830] RT-PCR of several RNA samples was used to analyze the expression of several Tas1b and Tas3a exchanges in several Col-0 cells. For RT-PCR, cDNA was generated using several non-specific primers for Tas1b and Tas3a via the qScript Flex cDNA Synthesis Kit (Quanta BioSciences). One cDNA reaction was performed on the sense strand, and another reaction was performed on the antisense strands of Tas1b and Tas3a. Several samples to be processed contained 165 ng / μl RNA.
[0831] For all RT-PCR reactions (same treatment, but using H2O instead of reverse transcriptase), a negative control without reverse transcriptase (-RT control) was used. This was to ensure that amplification in downstream PCR reactions did not occur due to DNA residue. A negative control with water was performed for each PCR reaction. A master mixture was prepared with RNA for each treatment's +RT / -RT. Additional premixes were prepared—(i) with reverse transcriptase and buffer (+RT) and (ii) with water and buffer (-RT) for all samples. Final primer concentration: 1 µM.
[0832] Primers: Tas1b - Tas1b has meaning: Tas1b_RT_A_R: 5´-TAACATAAAAATATTACAAATATCATTCCG-3´ (SEQ ID NO: 93) - Tas1b antonym: Tas1b_RT_B_F: 5´-TCAGAGTAGTTATGATTGATAGGATGG-3´ (SEQ ID NO: 94) These primers are used to process A, B, and E.
[0833] Tas3a - Tas3a has meaning: Tas3a_RT_A_R: 5´-GCTCAGGAGGGATAGACAAGG-3´ (SEQ ID NO: 95) - Tas3a antonym: Tas3a_RT_B_F: 5´-CTCGTTTTACAGATTCTATTCTATCTC-3´ (SEQ ID NO: 96) These primers are used to process C, D, and E.
[0834] PCR was performed on cDNA to detect the expression of Tas1b and Tas3a redirected to several nematode targets. To detect dsRNA transcribed from the Tas1b or Tas3a genes redirected to several target nematode genes, several PCR reactions were performed using the cDNA as a template, employing one nonspecific primer for Tas3a or Tas1b and another exchange-specific primer (i.e., binding only to the relevant Tas sequence that has been exchanged for several nucleotides after GEiGS-mediated redirection). The nonspecific primer annealing site was located slightly downstream of the sequence used to prepare the cDNA. The specific primer annealing site was located less than 200 bp downstream of the nonspecific primer annealing site. The expression of both strands of dsRNA was analyzed using the same method: sense and antisense. Several reactions were also performed on the -RT cDNA to ensure that residual DNA in the sample after DNase treatment was not amplified. As a negative control, each reaction was also performed on WT DNA to demonstrate that the amplification was Swap-specific. Each PCR reaction included an H2O negative control. 5 μL of each cDNA PCR reaction was used as a template.
[0835] Primers: Tas3a Sense has a sense chain-specific response: - Ribosomal protein 3a specificity: Tas3a RNA Nonspecific A F: 5´-TGACCTTGTAAGACCCCATCTC-3´ (SEQ ID NO: 97) Tas3a_RNA_Ribo3a30_Specificity_A_R: 5´-AggagaaaATTCGTAAGGTGAAGG-3´ (SEQ IDNO: 98) - WT specificity: Tas3a RNA Nonspecific A-F: 5´-TGACCTTGTAAGACCCCATCTC-3´ (SEQ ID NO: 99) Tas3a_RNA_WT_specific_A_R: 5´-GGTAGGAGAAAATGACTCGAACG-3´ (SEQ ID NO:100) Tas3a antisense strand specific response: - Ribosomal protein 3a specificity: Tas3a_RNA_Nonspecific_B_R: 5´-CAACCATACATCAATAACAAACAAAAG-3´ (SEQ ID NO:101) Tas3a_RNA_Ribo3a30_Specificity_B_F: 5´-ATATAGAATAGATatCGGCTTCTTCAG-3´ (SEQID NO: 102) - WT specificity: Tas3a_RNA_Nonspecific_B_R: 5´-CAACCATACATCAATAACAAACAAAAG-3´ (SEQ ID NO:103) Tas3a RNA Spliceo30 Specific B F: 5´-TCCTTTTTGACCAACATTTGTTTGT-3´ (SEQ ID NO: 104) Tas1b exhibits a sense chain-specific response: - Y25, COPI complex-specific β subunit: Tas1b RNA Nonspecific A F: 5´-GAGTCATTCATCGGTATCTAACC-3´ (SEQ ID NO:105) Tas1b RNA Y2530 Specific RNA: 5´-agCCGCCTCTGTGGATTCTTG-3´ (SEQ ID NO:106) - WT specificity: Tas1b RNA Nonspecific A F: 5´-GAGTCATTCATCGGTATCTAACC-3´ (SEQ ID NO:107) Tas1b RNA WT Specific A RR: 5´-TGGACTTAGAATATGCTATGTTGGAC-3´ (SEQ ID NO:108) Tas1b antisense strand specific response: - Y25, COPI complex-specific β subunit: Tas1b RNA Nonspecific BR: 5´-GCATATCCTAAAATATGTTTCGTTAAC-3´ (SEQ ID NO:109) Tas1b RNA Y2530 Specificity B F: 5´-TCGCCAAGAATCCACAGAGC-3´ (SEQ ID NO:110) - WT specificity: Tas1b RNA Nonspecific BR: 5´-GCATATCCTAAAATATGTTTCGTTAAC-3´ (SEQ ID NO:111) Tas1b_RNA_WT_Specificity_B_F: 5´-TAAGTCCAACATAGCATATTCTAAGTC-3´ (SEQ IDNO: 112) Study on the silencing activity of long dsRNA in *Nicotiana benthamiana* on TuMV plant materials Nicotiana bethamiana was grown in soil for 4 weeks under long-day conditions (16 hours of light, 8 hours of darkness) at 21°C until treatment.
[0836] TuMV-GFP vector cloning The TuMV-GFP cDNA cassette was amplified from the vector described in Touriño, A. et al. (Touriño, A., Sánchez, F., Fereres, A., and Ponz, F., 2008). High expression of several exogenous proteins in a biosafe viral vector is derived from turnip mosaic virus. (Spanish Journal of Agricultural Research, 6(S1), p. 48). Amplification was performed using primer sets 5'-ATGTTTGAACGATCGGGCCCaagggacacgaagtgatccg-3' (SEQ ID NO: 113) and 5'-CTCCACCATGTTCCCGGGggcacacagtgttcaacccc-3' (SEQ ID NO: 114). The amplicon was cloned into a binary vector containing the NPTII resistance gene, located in the T-DNA region, via a fusion reaction, according to the manufacturer's protocol. For the purpose of Agrobacterium infiltration, the vector was then transformed into Agrobacterium strain GV3101.
[0837] Agrobacterium induction and leaf infiltration 1. Agrobacterium is cultured in liquid form in LB medium.
[0838] 2. Centrifuge several cells and wash once with MMA medium (10 mM MES, 10 mM MgCl2 and 200 µM acetylsyl syringone, pH=5.6).
[0839] 3. Centrifuge several cells and remove the daughter cells (sub). Resuspend the particles in MMA medium until OD600 = 0.5.
[0840] 4. Gently shake the culture in the dark for 6 hours.
[0841] 5. As needed, combine several cultures (containing bacteria with several different vectors in a 1:1 ratio, each Agrobacterium containing a vector expressing a single gene). The final total Agrobacterium density - OD600 = 0.5. Add the TuMV-GFP vector to a final density of OD600 = 0.0001.
[0842] The induction cultures were infiltrated into several leaves of 4-week-old Nicotiana bonnet plants using a needle-free syringe.
[0843] Several gene sequences used for GEiGS-dsRNA silencing - AtTAS1B (At1g50055) – SEQ ID NO: 115 - GEiGS-TuMV – SEQ ID NO: 116 - GEiGS-TuMV-matured siRNA – SEQ ID NO: 117 - GEiGS-Virtual – SEQ ID NO: 118 - GEiGS-virtual-matured siRNA – SEQ ID NO: 119 -miR173_AT3G23125 – SEQ ID NO: 120 - miR173-mature miRNA – SEQ ID NO: 121 Study on the protection of Arabidopsis thaliana against TuMV infection and disease plant materials Several Arabidopsis seeds collected from several plants containing the desired GEiGS sequence were sterilized with chlorine and sown at 1 seed / well on several MS-S agar plates. Several two-week-old seedlings were transferred to soil. Several plants were grown at 24°C under several 16-hour light / 8-hour dark cycles. Unmodified (several plants) wild-type plants were grown and treated in parallel as controls.
[0844] Plant inoculation and analysis Several procedures for inoculating and analyzing plants with several TuMV vectors have been well established in the art and previously described in [Sardaru, P. et al., Molecular Plant Pathology (2018), 19:1984-1994]. Four-week-old Arabidopsis seedlings were inoculated with TuMV as previously described [Sánchez, F. et al., 1998, VirusResearch, 55(2): 207-219], or with TuMV-GFP as previously described [Touriño, A. et al., 2008, Spanish Journal of Agricultural Research, 6(S1), p. 48] to express several viral vectors. In the case of TuMV, symptom scoring occurred 10 to 28 days post-inoculation. In the case of TuMV-GFP, analysis of the GFP signal occurred 7 to 14 days post-inoculation.
[0845] In addition, 14 days post-inoculation, several new leaves growing above the inoculation site were harvested, and total RNA was extracted using a total RNA purification kit (Norgene Biotek Corp., Canada) according to the manufacturer's instructions. Small RNA analysis and RNA sequencing (RNA-seq) were performed to analyze gene expression and small RNA expression in these samples.
[0846] Research on tomato infection with whiteflies plant materials Several tomato plants were grown from seeds collected from several plants containing the desired GEiGS sequence, with one plant per pot grown at 22°C under several 16-hour light / 8-hour dark cycles. Unmodified (several plants) wild-type plants were grown and treated in parallel as a control.
[0847] Whitefly inoculation Five female whiteflies were introduced into a 4-week-old tomato plant. The whiteflies were placed in a cage with a leaf attached. Five days later, the number of dead and surviving whiteflies and eggs were counted.
[0848] In addition, infected leaves were harvested 5 days after inoculation, and total RNA was extracted. Dead and surviving whiteflies were collected separately, and total RNA was extracted from them. Small RNA analysis and RNA sequencing (RNA-seq) were performed to analyze gene expression and small RNA expression in these samples.
[0849] dsRNA research targeting genes in First-line worms nematodes Several plant-parasitic cyst nematodes, *Globoderarostochiensis* (pathogenetic type Ro1, from the James Hutton Institute Collection), are held at the University of Cambridge under DEFRA license 125034 / 359149 / 3. The nematodes are preserved on the potato cultivar *Solanum tuberosum* cultivar *Désirée*. Fifty cysts were mixed with a 50:50 sand:loam mixture in a 7-inch diameter pot. One tuber was planted in each pot and watered regularly for 3 months at 20°C. The plants were allowed to dry for 1 month, and then the cysts were collected from the soil using flotation followed by nested sieving. Several larvae hatched from the cysts by incubation with tomato root diffusion solution, with replacement every 2 to 3 days for up to 14 days. Several hatched larvae were stored in water containing 0.01% Tween-20 at 4 °C for up to one week before being used in several subsequent analyses.
[0850] Several sequences used - AtTAS3a_AT3G17185 – SEQ ID NO: 122 -GEiGS-ribosomal protein 3a-transcription- SEQ ID NO: 123 -GEiGS-ribosomal protein 3a-transcription- SEQ ID NO: 124 - represents a region homologous to the target gene designed via GEiGS to produce siRNA (i.e., the intended processed siRNA) in several nematodes. - GEiGS-Spliceosome SR protein transcript- SEQ ID NO: 125 - GEiGS-Spliceosome SR Protein Transcript- SEQ ID NO: 126 - Represents a region homologous to the target gene designed via GEiGS to produce siRNA (i.e., the intended processed siRNA) in several nematodes. -miR390_AT2G38325 – SEQ ID NO: 127 RNA preparation for feeding Total RNA was extracted from several permeated *N. benthamiana* leaves using Tri-Reagent (Sigma-Aldrich, USA), followed by two chloroform washes and overnight precipitation in isopropanol. The recovered RNA was further washed using standard sodium acetate precipitation.
[0851] Following the manufacturer's instructions, all recovered RNA was washed with Amicon® Ultra 0.5 mL Centrifugal Filter...
Claims
1. A method of producing a long dsRNA molecule in a plant cell capable of silencing a pest gene, comprising: The method comprises: (a) selecting a nucleic acid sequence in a plant genome, the nucleic acid sequence encoding a silencing molecule targeting a plant gene, the silencing molecule being capable of recruiting an RNA-dependent RNA polymerase RdRp; (b) modifying a nucleic acid sequence of the plant gene to confer a silencing specificity against the pest gene, such that a transcript of the plant gene comprising the silencing specificity base pairs with the silencing molecule capable of recruiting the RdRp to produce the long dsRNA molecule capable of silencing the pest gene, thereby producing the long dsRNA molecule in the plant cell capable of silencing the pest gene.
2. The method of claim 1, wherein: The silencing molecule capable of recruiting the RdRp comprises 21 to 24 nucleotides.
3. The method of any one of claims 1 to 2, wherein: The silencing molecule capable of recruiting the RdRp is selected from the group consisting of: trans-acting siRNA, phased small interfering RNA, microRNA, small interfering RNA, short hairpin RNA, Piwi-interacting RNA, transfer RNA, small nuclear RNA, ribosomal RNA, small nucleolar RNA, extracellular RNA, repeat-derived RNA, autonomous and non-autonomous transposable RNA.
4. The method of claim 3, wherein: The microRNA comprises a mature microRNA having 22 nucleotides.
5. The method of claim 3 or 4, wherein: The microRNA is selected from the group consisting of: miR-156a, miR-156c, miR-162a, miR-162b, miR-167d, miR-169b, miR-173, miR-393a, miR-393b, miR-402, miR-403, miR-447a, miR-447b, miR-447c, miR-472, miR-771, miR-777, miR-828, miR-830, miR-831, miR-831, miR-833a, miR-833a, miR-840, miR-845b, miR-848, miR-850, miR-853, miR-855, miR-856, miR-864, miR-2933a, miR-2933b, miR-2936, miR-4221, miR-5024, miR-5629, miR-5648, miR-5996, miR-8166, miR-8167a, miR-8167b, miR-8167c, miR-87e6187d, miR-8167f, miR-8177, and miR-8182.
6. The method of any one of claims 1 to 5, wherein: The plant gene is a non-protein-coding gene.
7. The method of any one of claims 1 to 6, wherein: The plant gene encodes a molecule having an intrinsic silencing activity against a native plant gene.
8. The method of any one of claims 1 to 7, wherein: The modification of step (b) comprises introducing a DNA editing agent into the plant cell, the DNA editing agent re-directing a silencing specificity of the plant gene to be against the pest gene, the pest gene and a native plant gene being different.
9. The method of claim 7 or 8, wherein: The plant gene having the intrinsic silencing activity is selected from the group consisting of: trans-acting siRNA, phased small interfering RNA, microRNA, small interfering RNA, short hairpin RNA, Piwi-interacting RNA, transfer RNA, small nuclear RNA, ribosomal RNA, small nucleolar RNA, extracellular RNA, autonomous and non-autonomous transposable RNA.
10. The method of any one of claims 7 to 9, wherein: The plant gene having the intrinsic silencing activity encodes a phased secondary siRNA producing molecule.
11. The method of any one of claims 7 to 9, wherein: The plant gene having the intrinsic silencing activity is a trans-acting siRNA producing molecule.
12. The method of any one of claims 1 to 11, wherein: The silencing specificity of the plant gene is determined by measuring a transcription level of the pest gene.
13. The method of any one of claims 1 to 12, wherein: The silencing specificity of the plant gene is determined phenotypically.
14. The method of claim 13, wherein: The determination phenotypically is achieved by determining pest resistance of the plant.
15. The method of any one of claims 1 to 14, wherein: The silencing specificity of the plant gene is determined genotypically.
16. The method of claim 15, wherein: A plant phenotype is determined prior to a plant genotype.
17. The method of claim 15, wherein: A plant genotype is determined prior to a plant phenotype.
18. A method of producing a long dsRNA molecule in a plant cell capable of silencing a pest gene, comprising: The method comprises: (a) selecting a nucleic acid sequence of a plant gene, the nucleic acid sequence of the plant gene showing a predetermined sequence homology with a nucleic acid sequence of the pest gene; (b) modifying a plant endogenous nucleic acid sequence encoding an RNA molecule to confer silencing specificity to the plant gene, such that a number of small RNA molecules processed from the RNA molecule to produce the long dsRNA molecule capable of silencing the pest gene, capable of base complementing a transcript of the plant gene, are able to recruit RNA-dependent RNA polymerase RdRp, thereby producing the long dsRNA molecule in the plant cell capable of silencing the pest gene.
19. The method of claim 18, wherein: The predetermined sequence homology comprises 75 to 100% identity.
20. The method of any one of claims 18-19, wherein: The small RNA molecule capable of recruiting the RdRp comprises 21 to 24 nucleotides.
21. The method of any one of claims 18 to 20, wherein: The small RNA molecule capable of recruiting the RdRp is selected from the group consisting of: microRNA, small interfering RNA, short hairpin RNA (shRNA), Piwi-interacting RNA, trans-acting siRNA, phased small interfering RNA, transfer RNA, small nuclear RNA, ribosomal RNA, small nucleolar RNA, extracellular RNA, repeat-derived RNA, autonomous and non-autonomous transposable RNA.
22. The method of any one of claims 18 to 21, wherein: The RNA molecule has an intrinsic silencing activity against a native plant gene.
23. The method of any one of claims 18 to 22, wherein: The modification of step (b) comprises introducing into the plant cell a DNA editing agent that redirects the silencing specificity of the RNA molecule to the plant gene, the plant gene and a native plant gene being different.
24. The method of any one of claims 18 to 23, wherein: The plant gene showing the predetermined sequence homology with the nucleic acid sequence of the pest gene does not encode a silencing molecule.
25. The method of any one of claims 18 to 24, wherein: The silencing specificity of the RNA molecule is determined by measuring a transcription level of the plant gene or the pest gene.
26. The method of any one of claims 18 to 25, wherein: The silencing specificity of the RNA molecule is phenotypically determined.
27. The method of claim 26, wherein: The phenotypically determined is achieved by determining a pest resistance of the plant.
28. The method of any one of claims 18 to 27, wherein: The silencing specificity of the RNA molecule is genotypically determined.
29. The method of claim 28, wherein: A plant phenotype is determined prior to a plant genotype.
30. The method of claim 28, wherein: A plant genotype is determined prior to a plant phenotype.
31. The method of any one of claims 8 to 17 or 23 to 30, wherein: The DNA editing agent comprises at least one sgRNA.
32. The method of any one of claims 8 to 17 or 23 to 31, wherein: The DNA editing agent does not comprise an endonuclease.
33. The method of any one of claims 8-17 or 23-31, wherein: The DNA editing agent comprises an endonuclease.
34. The method of any one of claims 8-17 or 23-33, wherein: The DNA editing agent is a DNA editing system selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, and a CRISPR-endoclease, a dCRISPR-endonuclease, and a homing endonuclease.
35. The method of claim 33 or 34, wherein: The endonuclease comprises Cas9.
36. The method of any one of claims 8 to 17 or 23 to 35, wherein: The DNA editing agent is applied to the cell in the form of DNA, RNA, or RNP.
37. The method of any one of claims 1 to 36, wherein: The plant cell is a protoplast.
38. The method of any one of claims 1 to 37, wherein: A dsRNA molecule is processed by a cellular RNAi processing machinery.
39. The method of any one of claims 1 to 38, wherein: A dsRNA molecule is processed into secondary small RNAs.
40. The method of any one of claims 1 to 39, wherein: The dsRNA and / or the secondary small RNAs comprise a silencing specificity for a pest gene.
41. A method of producing a pest tolerant or pest resistant plant comprising: The method comprises producing a long dsRNA molecule in a plant cell, the long dsRNA molecule in the plant cell being capable of silencing a pest gene according to any one of claims 1 to 40.
42. The method of claim 41, wherein: The pest is an invertebrate.
43. The method of claim 41 or 42, wherein: The pest is selected from the group consisting of a virus, an ant, a termite, a bee, a wasp, a caterpillar, a cricket, a locust, a beetle, a snail, a slug, a nematode, a bed bug, a fly, a fruit fly, a whitefly, a mosquito, a grasshopper, a planthopper, a earwig, an aphid, a scale, a thrips, a spider, a mite, a psyllid, a tick, a moth, a worm, a scorpion, and a fungus.
44. A plant, comprising: The plant is produced by the method according to any one of claims 1 to 43.
45. The plant of claim 44, wherein: The plant is selected from the group consisting of a crop, a flowering plant, a weed, and a tree.
46. The plant of claim 44 or 45, wherein: The plant is non-transgenic.
47. A cell, comprising: The cell is from a plant according to any one of claims 44 to 46.
48. A seed, wherein: The seed is from a plant according to any one of claims 44 to 46.
49. A method for producing pest-tolerant or pest-resistant plants, characterized in that: The method comprises: (a) breeding a plant according to any one of claims 44 to 46; and (b) selecting progeny plants that express the long dsRNA molecule capable of inhibiting the pest gene and that do not comprise the DNA editing agent, thereby producing the pest-resistant or pest-tolerant plant.
50. A method of producing a plant or plant cell of any one of claims 44 to 47, comprising: The method comprises culturing the plant or plant cell under conditions that allow for propagation.
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