Introducing silencing activity into dysfunctional RNA molecules and altering their specificity for genes of interest

Abstract

Techniques are provided for conferring silencing activity to silencing-dysfunctional RNA molecules (eg, miRNA-like molecules) in eukaryotic cells. [Solution] A method for generating an RNA molecule having silencing activity in a cell is provided, comprising: (a) identifying a nucleic acid sequence that does not contain complete identity to a nucleic acid sequence encoding an RNA molecule that associates with RISC; (b) selecting a transcribable nucleic acid sequence that encodes the RNA molecule; (c) selecting a transcribable nucleic acid sequence that encodes an aberrantly processed RNA molecule that exhibits a predetermined range of sequence homology; and (d) associating with RISC and conferring processivity to a small RNA that is complementary to a first target RNA or a target RNA of interest.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority from UK Patent Application No. 1903519.5 filed on 14 March 2019, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Description An ASCII file entitled 81320 Sequence Listing.txt, containing 221,283 bytes and created on March 12, 2020, which was filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] FIELD OF THE INVENTION AND BACKGROUND ART In some embodiments, the present invention relates to conferring silencing activity to silencing-dysfunctional RNA molecules (e.g., miRNA-like molecules) in eukaryotic cells, and in some cases altering the silencing specificity of the RNA molecules for silencing an endogenous or exogenous target RNA of interest. [Background technology]

[0004] Recent advances in genome editing technology have made it possible to alter DNA sequences in living cells by editing just a few of the billions of nucleotides in their genomes. Over the past decade, the tools and expertise required for using genome editing in human somatic and pluripotent cells have increased, to the point where this approach is now widely deployed as a strategy for treating human diseases. The basic process relies on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to repair the break (e.g., by non-homologous end joining (NHEJ) or homologous recombination (HR), the latter of which allows precise nucleotide changes to be made to a DNA sequence using an exogenously provided donor template [Porteus, Annu Rev Pharmacol Toxicol. (2016) 56:163-90].

[0005] For example, for potential therapeutics, three major approaches use mutagenic genome editing (NHEJ) in cells: (a) knocking out functional genetic elements by causing spatially precise insertions or deletions, (b) creating insertions or deletions that compensate for the underlying frameshift mutation, reactivating partially functional or nonfunctional genes, and (c) creating defined gene deletions. While NHEJ editing is used in several different applications, genome editing by homologous recombination (HR) is likely to offer the broadest range of applications because HR is a rare event but highly precise, relying on an exogenously provided template to copy specific, predetermined sequences during the repair process.

[0006] Currently, the four main applications of HR-mediated genome editing are (a) gene correction (i.e., correcting diseases caused by point mutations in a single gene), (b) functional gene correction (i.e., correcting diseases caused by mutations scattered throughout a gene), (c) safe harbor gene addition (i.e., when precise regulation is not required or non-physiological levels of the transgene are desired), and (d) targeted transgene addition (i.e., when precise regulation is required) [Porteus (2016), op. cit.].

[0007] Genome compilation of RNA molecules in various eukaryotic organisms (e.g., mouse, human, shrimp, and plants) Previous studies on miRNAs have focused on, for example, knocking out the activity of miRNA genes or altering their binding sites in target RNAs.

[0008] Regarding genome editing in human cells, Jiang et al. [Jiang et al., RNA Biology (2014) 11(10):1243-9] used CRISPR / Cas9 to delete human miR-93 from the cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region, including the Drosha processing site (i.e., the site where Drosha, a double-stranded RNA-specific RNase III enzyme, binds to and cleaves the primary miRNA (pri-miRNA) in the host cell nucleus, thereby processing it into pre-miRNA) and the seed sequence (i.e., a conserved heptametric sequence essential for miRNA binding to mRNA, typically located 2-7 positions from the 5' end of the miRNA). According to Jiang et al., even a single-nucleotide deletion completely knocked out the targeted miRNA with high specificity.

[0009] Regarding genome editing in mouse species, Zhao et al. [Zhao et al., Scientific Reports (2014) 4:3943] provided a strategy for miRNA inhibition using the CRISPR-Cas9 system in mouse cells. Zhao used a specially designed sgRNA to cleave the miRNA gene at a single site with Cas9 nuclease, resulting in the knockout of the miRNA in these cells.

[0010] Regarding genome editing in plants, Bortesi and Fischer [Bortesi and Fischer, Biotechnology Advances (2015) 33:41-52] discuss the use of CRISPR-Cas9 technology in plants in comparison with ZFNs and TALENs, and Basak and Nithin [Basak and Nithin, Front Plant Sci. (2015) 6:1001] teach that CRISPR-Cas9 technology has been applied to knockdown protein-encoding genes in model plants such as Arabidopsis and tobacco, as well as in crops including wheat, maize, and rice.

[0011] In addition to disrupting miRNA activity or target binding sites, gene silencing has been achieved using artificial miRNAs (amiRNAs) to silence endogenous and exogenous target genes [Tiwari et al. Plant Mol Biol (2014) 86:1]. Similar to miRNAs, amiRNAs are single-stranded, approximately 21 nucleotides (nt) long, and are engineered by replacing the double-stranded mature miRNA sequence within a pre-miRNA [Tiwari et al. (2014) pp. 113-114]. These amiRNAs are introduced as transgenes within artificial expression cassettes (containing promoters, terminators, etc.) [Carbonell et al., Plant Physiology (2014) pp. 113-1149] and are processed via the small RNA biogenesis and silencing machinery to downregulate target expression. According to Schwab et al. [Schwab et al. The 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 plants, especially when several related but non-identical target genes need to be downregulated.

[0012] Senis et al. [Senis et al., Nucleic Acids Research (2017) Vol. 45(1): e3] disclose the introduction of a promoterless antiviral RNAi hairpin into an endogenous miRNA locus. Specifically, Senis et al. introduce a sequence-specific nuclease, such as Cas9 or TALEN nuclease, into the miRNA locus. In this approach, an amiRNA precursor transgene (hairpin pri-amiRNA) is inserted adjacent to a naturally occurring miRNA gene (e.g., miR122) via homology-dependent DNA recombination. This approach utilizes transcriptionally active DNA expressing a natural miRNA (miR122) to use a promoter- and terminator-less amiRNA; i.e., the endogenous promoter and terminator drive and regulate transcription of the inserted amiRNA transgene.

[0013] Various methods for introducing RNA and / or proteins into cells without using DNA have already been reported. For example, RNA transfection using electroporation and lipofection is described in U.S. Patent Application Publication No. 20160289675. Cho [Cho et al., "Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins," Genetics (2013) 195:1177-1180] described direct delivery of Cas9 / sgRNA ribonucleoprotein (RNP) complexes into cells by microinjection of Cas9 protein and sgRNA complexes. Kim [Kim et al., "Highly efficient RNA-guided genome editing in human cells via delivery Delivery of Cas9 protein / sgRNA complexes via electroporation has been described by Zuris [Zuris et al., “Cationic lipid-mediated [Delivery of proteins enables efficient protein-based genome editing in vitro and in vivo] reported liposome-mediated delivery of Cas9 protein-associated sgRNA complexes. Summary of the Invention

[0014] According to some embodiments of the present invention, there is provided a method for generating RNA molecules having silencing activity in a cell, comprising: (a) identifying nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology, not including complete identity, to a nucleic acid sequence encoding an RNA molecule that associates with an RNA-induced silencing complex (RISC); (b) determining transcription of the nucleic acid sequences encoding the RNA molecules to select a transcribable nucleic acid sequence encoding the RNA molecule that exhibits the predetermined range of sequence homology; and (c) determining transcription of the nucleic acid sequences encoding the transcribable nucleic acid sequence encoding the RNA molecule that exhibits the predetermined range of sequence homology. (d) modifying the nucleic acid sequence of the transcribable nucleic acid sequence encoding the aberrantly processed RNA molecule exhibiting the predetermined sequence homology range to confer processivity into small RNA that associates with RISC and is complementary to a first target RNA, thereby generating an RNA molecule with silencing activity in a cell.

[0015] According to aspects of some embodiments of the present invention, there is provided a genetically modified cell comprising a genome comprising a polynucleotide sequence encoding an RNA molecule having a nucleic acid sequence alteration that results in the RNA molecule being processed into a small RNA that associates with RISC, wherein the processing of the RNA molecule is absent in a wild-type cell of the same origin that does not have the nucleic acid sequence alteration.

[0016] According to an aspect of some embodiments of the present invention there is provided a plant cell produced according to the method of some embodiments of the present invention.

[0017] According to an aspect of some embodiments of the present invention there is provided a plant comprising a plant cell of some embodiments of the present invention.

[0018] According to an aspect of some embodiments of the present invention, there is provided a method of generating a plant with reduced expression of a target gene, the method comprising: (a) breeding a plant of some embodiments of the present invention; and (b) selecting progeny plants with reduced expression of the target RNA of interest, or progeny that comprise a silencing RNA molecule specific for the target RNA of interest and that do not contain a DNA editing agent, thereby generating a plant with reduced expression of the target gene.

[0019] According to an aspect of some embodiments of the present invention, there is provided a method of generating a plant comprising an RNA molecule having silencing activity against a target RNA of interest, the method comprising: (a) breeding a plant of some embodiments of the present invention; and (b) selecting progeny plants comprising the RNA molecule having silencing activity against the target RNA of interest, or progeny comprising silencing specificity in the RNA molecule for the target RNA of interest and not comprising a DNA editing agent, thereby generating a plant comprising the RNA molecule having silencing activity against the target RNA of interest.

[0020] According to an aspect of some embodiments of the present invention there is provided a method of producing a plant or plant cell of some embodiments of the present invention comprising growing the plant or plant cell under conditions that allow propagation.

[0021] According to an aspect of some embodiments of the present invention there is provided a seed of a plant of some embodiments of the present invention or a plant produced by some embodiments of the present invention.

[0022] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease in a subject in need thereof, the method comprising generating an RNA molecule having silencing activity and / or specificity according to the method of some embodiments of the present invention, wherein the RNA molecule has silencing activity for a transcript of a gene associated with the onset or progression of the disease, thereby treating the subject.

[0023] According to an aspect of some embodiments of the present invention there is provided a method of introducing silencing activity into a first RNA molecule in a cell, comprising: (a) i. the first nucleic acid sequence is transcribed within the cell into a first RNA molecule; ii. the sequence of the first RNA molecule has partial homology, except for sequence identity, to the sequence of a second RNA molecule, the second RNA molecule being processible into a third RNA molecule having silencing activity, the second RNA molecule being encoded by a second nucleic acid sequence in the cell; and iii. selecting a first nucleic acid sequence in the cell, wherein the first RNA molecule is either unprocessable or processable differently from a second RNA molecule, such that the first RNA molecule is not processed into an RNA molecule having the same properties of silencing activity as a third RNA molecule; (b) modifying the first nucleic acid sequence to encode a modified first RNA molecule, wherein the modified first RNA molecule is processible into a fourth RNA molecule in the same manner as the second RNA molecule is processible into the third RNA molecule, such that the fourth RNA molecule has the same qualitative silencing activity as the third RNA molecule; thereby introducing silencing activity into said first RNA molecule; A method is provided which includes:

[0024] According to some embodiments of the invention, the RNA molecules of step (a) encoded by the identified nucleic acid sequences exhibit a predetermined range of sequence homology, which does not include complete identity, to RNA molecules that associate with RISC and / or are processed into molecules that associate with RISC.

[0025] According to some embodiments of the invention, conferring processivity in step (d) comprises conferring standard processing to an RNA molecule encoded by a nucleic acid sequence encoding an RNA molecule that associates with an RNA-induced silencing complex (RISC).

[0026] According to some embodiments of the invention, the method further comprises determining the genomic location of nucleic acid sequences encoding RNA molecules that exhibit the predetermined sequence homology range of step (a).

[0027] According to some embodiments of the invention, the genomic location is within a non-coding gene.

[0028] According to some embodiments of the invention, the genomic location is within an intron of a non-coding gene.

[0029] According to some embodiments of the invention, the genomic location is within a coding gene.

[0030] According to some embodiments of the invention, the genomic location is within an exon of a coding gene.

[0031] According to some embodiments of the invention, the genomic location is within an exon encoding an untranslated region (UTR) of a coding gene.

[0032] According to some embodiments of the invention, the genomic location is within an intron of the coding gene.

[0033] According to some embodiments of the invention, the RNA molecule is encoded by a nucleic acid sequence located in a non-coding gene.

[0034] According to some embodiments of the invention, the RNA molecule is encoded by a nucleic acid sequence located in a coding gene.

[0035] According to some embodiments of the invention, the RNA molecule is encoded by a nucleic acid sequence located within an exon of the encoding gene.

[0036] According to some embodiments of the invention, the RNA molecule is encoded by a nucleic acid sequence located within an exon encoding an untranslated region (UTR) of the coding gene.

[0037] According to some embodiments of the invention, the RNA molecule is encoded by a nucleic acid sequence located within an intron of the encoding gene.

[0038] According to some embodiments of the invention, the genomic location is within an intron of a non-coding gene.

[0039] According to some embodiments of the invention, the sequence homology range comprises 75% to 99.6% identity to a nucleic acid sequence encoding an RNA molecule that associates with RISC.

[0040] According to some embodiments of the invention, steps (b) and / or (c) are affected by aligning the small RNA expression data to the genome of the cell and determining the amount of reads that map to each genomic location.

[0041] According to some embodiments of the invention, the alignment of the small RNA is to a predetermined location in the genome of the cell without any mismatches.

[0042] According to some embodiments of the present invention, the nucleic acid sequence of a transcribable nucleic acid sequence is modified to confer a structure to an aberrantly processed RNA molecule, such that the RNA molecule is processed into small RNAs that associate with RISC.

[0043] According to some embodiments of the invention, nucleic acid sequence modifications of transcribable nucleic acid sequences encoding aberrantly processed RNA molecules that exhibit a predetermined sequence homology range are made in nucleic acids other than those corresponding to the binding site for the first target RNA.

[0044] According to some embodiments of the invention, the processivity is provided by a cellular nuclease selected from the group consisting of Dicer, Argonaute, tRNA cleavage enzymes, and Piwi-binding RNA (piRNA) associated proteins.

[0045] According to some embodiments of the invention, the modification in step (d) comprises introducing into the cell a DNA editing agent that reactivates the silencing activity of the aberrantly processed RNA molecule toward the first target RNA, thereby generating an RNA molecule with silencing activity in the cell.

[0046] According to some embodiments of the invention, the method further includes altering the specificity of the RNA molecule having silencing activity in the cell, the method comprising introducing into the cell a DNA editing agent that redirects the silencing specificity of the RNA molecule toward a target RNA of interest that is different from the first target RNA, thereby altering the specificity of the RNA molecule having silencing activity in the cell.

[0047] According to some embodiments of the invention, the method further includes altering the specificity of the RNA molecule having silencing activity in the cell, wherein the DNA editing agent redirects the silencing specificity of the RNA molecule toward a target RNA of interest that is different from the first target RNA, thereby altering the specificity of the RNA molecule having silencing activity in the cell.

[0048] According to some embodiments of the invention, the method further includes altering the specificity of the RNA molecule having silencing activity in the cell, the method comprising introducing into the cell a DNA editing agent that redirects the silencing specificity of the RNA molecule toward a target RNA of interest that is different from the first target RNA, thereby altering the specificity of the RNA molecule having silencing activity in the cell.

[0049] According to some embodiments of the invention, the nucleic acid sequence encoding the RNA molecule identified in step (a) is homologous to a gene encoding a silencing RNA molecule whose silencing activity and / or processing into small silencing RNAs depends on its secondary structure.

[0050] According to some embodiments of the present invention, the nucleic acid sequence encoding the RNA molecule of step (a) The strings are homologous to genes encoding miRNA precursors.

[0051] According to some embodiments of the invention, the silencing RNA molecule, whose silencing activity and / or processing into small silencing RNA depends on secondary structure, is selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA or U-RNA), small nucleolar RNA (snoRNA), small Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), RNA from repeats, RNA from autonomous and non-autonomous transposable and retrotransposable elements, RNA of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNA (lncRNA).

[0052] According to some embodiments of the present invention, the processing is standard processing.

[0053] According to some embodiments of the invention, the RNA molecule has silencing activity.

[0054] According to some embodiments of the invention, the RNA molecule is selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-binding RNA (piRNA), phased small interfering RNA (phasiRNA), trans-acting siRNA (tasiRNA), transfer RNA fragments (tRF), small nuclear RNA (snRNA), RNA of transposable and / or retrotransposable origin, autonomous and non-autonomous transposable and / or retrotransposable RNA.

[0055] According to some embodiments of the invention, the method further comprises introducing a donor oligonucleotide into the cell.

[0056] According to some embodiments of the invention, the DNA editing agent comprises at least one sgRNA.

[0057] According to some embodiments of the invention, the DNA editing agent does not comprise an endonuclease.

[0058] According to some embodiments of the invention, the DNA editing agent comprises an endonuclease.

[0059] According to some embodiments of the invention, the DNA editing agent is a DNA editing system selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR endonucleases, dCRISPR endonucleases, and homing endonucleases.

[0060] According to some embodiments of the invention, the endonuclease comprises Cas9.

[0061] According to some embodiments of the invention, the DNA editing agent is applied to the cell as DNA, RNA, or RNP.

[0062] According to some embodiments of the invention, the DNA editing agent is linked to a reporter to monitor its expression in a cell.

[0063] According to some embodiments of the invention, the reporter is a fluorescent protein.

[0064] According to some embodiments of the invention, the target RNA of interest is endogenous to the cell.

[0065] According to some embodiments of the invention, the target RNA of interest is exogenous to the cell.

[0066] According to some embodiments of the present invention, the silencing specificity of an RNA molecule is determined by measuring the RNA or protein levels of the target RNA of interest.

[0067] According to some embodiments of the present invention, the silencing specificity of an RNA molecule is determined phenotypically.

[0068] According to some embodiments of the present invention, the specificity of the RNA molecule is determined phenotypically by determining at least one phenotype selected from the group consisting of cell size, rate / inhibition of growth, cell shape, cell membrane integrity, tumor size, tumor shape, organism coloration, organism size, crop yield, metabolic profile, fruit traits, biotic stress resistance, abiotic stress resistance, infection parameters, and inflammation parameters.

[0069] According to some embodiments of the present invention, the silencing specificity of an RNA molecule is determined genotypically.

[0070] According to some embodiments of the invention, the cell is a eukaryotic cell.

[0071] According to some embodiments of the invention, the eukaryotic cell is obtained from a eukaryotic organism selected from the group consisting of plants, mammals, invertebrates, insects, nematodes, birds, reptiles, fish, crustaceans, fungi, and algae.

[0072] According to some embodiments of the invention, the eukaryotic cell is a plant cell.

[0073] According to some embodiments of the invention, the plant cell is a protoplast.

[0074] According to some embodiments of the invention, the plant is non-transgenic.

[0075] According to some embodiments of the invention, the plant is a transgenic plant.

[0076] According to some embodiments of the present invention, the plants are not genetically modified (non-GMO).

[0077] According to some embodiments of the present invention, the plants are genetically modified (GMO).

[0078] According to some embodiments of the invention, breeding comprises crossing or selfing.

[0079] According to some embodiments of the invention, the eukaryotic cell is a non-human animal cell.

[0080] According to some embodiments of the invention, the eukaryotic cell is a non-human mammalian cell.

[0081] According to some embodiments of the invention, the eukaryotic cell is a human cell.

[0082] According to some embodiments of the present invention, the nucleic acid sequence encoding the RNA molecule is selected from the group consisting of the nucleic acid sequences set forth in any of SEQ ID NOs: 352-392.

[0083] According to some embodiments of the invention, the eukaryotic cell is a totipotent stem cell.

[0084] According to some embodiments of the present invention, the gene associated with the onset or progression of the disease is a gene encoding a pathogen. Contains genes.

[0085] According to some embodiments of the invention, the gene associated with the onset or progression of the disease comprises a gene of the subject.

[0086] According to some embodiments of the invention, the disease is selected from the group consisting of an infectious disease, a monogenic recessive genetic disorder, an autoimmune disease, and a cancerous disease.

[0087] According to some embodiments of the invention, the second RNA molecule is an RNA molecule having a secondary structure that allows it to be processed into an RNA having silencing activity, optionally wherein the silencing activity is mediated through association with RISC.

[0088] According to some embodiments of the present invention, the RNA molecule having a secondary structure that allows it to be processed into an RNA with silencing activity is selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA or URNA), small nucleolar RNA (snoRNA), small Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), RNA from repeats, RNA from autonomous and non-autonomous transposable and retrotransposable elements, RNA of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNA (lncRNA).

[0089] According to some embodiments of the invention, the first nucleic acid sequence provides a secondary structure that allows the modified first RNA molecule to be processed into a fourth RNA molecule.

[0090] According to some embodiments of the invention, modifying the first nucleic acid sequence comprises modifying the sequence such that the modified first RNA molecule has essentially the same secondary structure as the second RNA molecule.

[0091] According to some embodiments, the secondary structure is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to the secondary structure of a second RNA molecule (e.g., when the secondary structure of a first RNA molecule is translated into a linear string form and compared to the string form of the secondary structure of a second RNA molecule).

[0092] According to some embodiments of the present invention, the first nucleic acid molecule is a gene derived from human (H. sapiens) and is a gene selected from the group consisting of genes having a sequence set forth in any one of SEQ ID NOs: 352 to 392.

[0093] According to some embodiments of the invention, the subject is a human subject.

[0094] 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [Brief explanation of the drawings]

[0095] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made specifically to the drawings in detail, stressing that the features shown are by way of example and are intended to provide an illustrative discussion of embodiments of the invention, In this regard, the accompanying description of the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced.

[0096] In the figure, [Figure 1] Figure 1 is a flowchart of an embodiment of a computational pipeline for conferring silencing activity and redirecting the silencing specificity of dysfunctional non-coding RNA molecules. Note that the computational genome editing-induced gene silencing (GEiGS) pipeline applies biological metadata to enable the automated generation of GEiGS DNA templates that are used to minimally edit miRNA genes to de novo gain-of-function, i.e., redirect their silencing ability against target sequences of interest. [Figure 2]Figure 2 is a photograph showing the miRbase representation of smallRNAseq profiling of functional miRNAs. Note the difference in detection of the two mature miRNA strands. The miRNA with high read counts is typically functional (the guide strand), while the other, with few or no read counts, is typically degraded in the cell (the passenger strand). However, in some cases, both strands of a mature miRNA are functional (each targeting a different transcript). [Figure 3] Figure 3 is a graph showing the number of RNA-seq reads covering miRNA-like sequences. The x-axis represents the miRNA-like sequences expressed in different species. The y-axis represents the number of different RNA-seq reads covering the miRNA-like sequences. "has" stands for human, "ath" for Arabidopsis thaliana, and "cel" for C. elegans. [Figure 4] Figure 4 is a flowchart of an embodiment of a computational pipeline for generating GEiGS templates. The computational GEiGS pipeline applies biological metadata and allows for the automated generation of GEiGS DNA donor templates that are used to minimally edit endogenous non-coding RNA genes (e.g., miRNA genes) to de novo gain-of-function, i.e., redirect their silencing capacity to target gene expression of interest. [Figure 5]Figure 5 is a flowchart of an embodiment of genome editing-induced gene silencing (GEiGS), which replaces endogenous miRNAs with siRNAs targeting PDS genes, thereby inducing gene silencing of the endogenous PDS genes. A two-component system is used to introduce the modifications. First, the CRISPR / CAS9 system cleaves the selected locus through a specific guide RNA designed in a GFP-containing vector, promoting homologous DNA repair (HDR) at that site. Second, a DONOR sequence, which modifies the miRNA sequence as desired to target the newly assigned gene, is introduced as a template for HDR. This system is used to transform protoplasts, which are then enriched by FACS due to the GFP signal of the CRISPR / CAS9 vector, recovered, and regenerated into plants. [Figure 6A-C] Figures 6A-C are photographs showing that silencing the PDS gene causes photobleaching. Silencing the PDS gene in Nicotiana (Figures 6A-B) and Arabidopsis (Figure 6C) plants causes photobleaching in N. benthamiana (Figure 6B) and Arabidopsis (Figure 6C, right). Photographs were taken 3.5 weeks after PDS silencing. [Figure 7] FIG. 7 provides a schematic diagram of an embodiment of a process for reactivating or redirecting silencing activity in an RNA transcript according to the present invention. [Figure 8A-B] 8A-B provide schematic diagrams of vectors used to transfect Arabidopsis protoplasts, as described in Example 2 herein below, to test the processivity and silencing activity of (FIG. 8A) a precursor of a wild-type miRNA, a precursor of a "Dead" miRNA-like molecule, and a precursor of a "Dead" miRNA-like molecule with reactivated silencing activity, and (FIG. 8B) a precursor of a "Dead" miRNA-like molecule with reactivated silencing activity and a precursor of a "Dead" miRNA-like molecule with silencing activity redirected to target the PDS3 gene. [Figure 9A-1]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9A-2]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9B]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9C-D]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9E]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9F]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 9G-H]Figures 9A–H provide the following: (Figure 9A) Schematic representations of the predicted secondary structures of the following Arabidopsis precursors encoded by the following miRNAs or miRNA-like genes: wild-type miR405a, miRNA-like miR859_Dead, miRNA-like miR859_Dead with reactivated silencing activity (miR859_reactivated), and miRNA-like miR859_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR859_redirected). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9B. (Figure 9C) and (Figure 9D) are bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9A). Dark bars represent experimental treatments, and light bars represent the respective controls. p values ​​in parentheses in the graphs are from Student's t-test, and error bars represent standard error. (Figure 9E) Schematic representation of the predicted secondary structures of the following Arabidopsis precursors encoded by the miRNA or miRNA-like genes: wild-type miR8174, miRNA-like miR1334_Dead, miRNA-like miR1334_Dead with reactivated silencing activity (miR1334_Reactivated), and miRNA-like miR1334_Dead with activated silencing activity and redirected silencing activity toward the PDS3 gene (miR1334_Redirect). The gray boxes in each structure mark the guide strand at the corresponding position in the mature miRNA or miRNA-like precursor. The alignment of each guide strand to its target sequence is further presented in Figure 9F. Bar graphs comparing the silencing activity (measured by the reduction in the ratio between luciferase (LUC) and normalized fluorescent protein (FP)) observed when Arabidopsis protoplasts were transfected with vectors expressing the vectors shown in (Figure 9G) and (Figure 9H) (Figure 9E).Dark bars represent experimental treatments and light bars represent the respective controls; p values ​​given in parentheses in the graphs are from Student's t-test and error bars represent standard errors. [Figure 10A-D] Figures 10A–N provide plots of the small RNA distribution and secondary structure of the Arabidopsis miRNA-like gene ath_dead_mir1334 and its corresponding WT miRNA ath-mir-8174 (MI0026804). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing smallRNAseq reads of all sizes. Read counts were normalized to RPKM, and plots were created for specific size groups if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The secondary structure of each precursor sequence was generated using the RNAplot module of the ViennaRNA package. Specifically, Figure 10A shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the WT precursor sequence (miRNA gene ath-mir-8174, located at ch3 positions 16589414–16589527). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 10G shows the secondary structure of the previously described WT miRNA precursor. Figure 10H shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the mir-like gene precursor sequence located at ch5 positions 13644905–1364500. Figure 10N shows the secondary structure of the mir-like precursor ath_dead_mir1334. [Figure 10E-H]Figures 10A–N provide plots of the small RNA distribution and secondary structure of the Arabidopsis miRNA-like gene ath_dead_mir1334 and its corresponding WT miRNA ath-mir-8174 (MI0026804). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing smallRNAseq reads of all sizes. Read counts were normalized to RPKM, and plots were created for specific size groups if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The secondary structure of each precursor sequence was generated using the RNAplot module of the ViennaRNA package. Specifically, Figure 10A shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the WT precursor sequence (miRNA gene ath-mir-8174, located at ch3 positions 16589414–16589527). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 10G shows the secondary structure of the previously described WT miRNA precursor. Figure 10H shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the mir-like gene precursor sequence located at ch5 positions 13644905–1364500. Figure 10N shows the secondary structure of the mir-like precursor ath_dead_mir1334. [Figure 10I-L]Figures 10A–N provide plots of the small RNA distribution and secondary structure of the Arabidopsis miRNA-like gene ath_dead_mir1334 and its corresponding WT miRNA ath-mir-8174 (MI0026804). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing smallRNAseq reads of all sizes. Read counts were normalized to RPKM, and plots were created for specific size groups if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The secondary structure of each precursor sequence was generated using the RNAplot module of the ViennaRNA package. Specifically, Figure 10A shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the WT precursor sequence (miRNA gene ath-mir-8174, located at ch3 positions 16589414–16589527). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 10G shows the secondary structure of the previously described WT miRNA precursor. Figure 10H shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the mir-like gene precursor sequence located at ch5 positions 13644905–1364500. Figure 10N shows the secondary structure of the mir-like precursor ath_dead_mir1334. [Figure 10M-N]Figures 10A–N provide plots of the small RNA distribution and secondary structure of the Arabidopsis miRNA-like gene ath_dead_mir1334 and its corresponding WT miRNA ath-mir-8174 (MI0026804). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing smallRNAseq reads of all sizes. Read counts were normalized to RPKM, and plots were created for specific size groups if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The secondary structure of each precursor sequence was generated using the RNAplot module of the ViennaRNA package. Specifically, Figure 10A shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the WT precursor sequence (miRNA gene ath-mir-8174, located at ch3 positions 16589414–16589527). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 10G shows the secondary structure of the previously described WT miRNA precursor. Figure 10H shows a distribution plot of all 20-bp small RNA-seq reads from roots that perfectly matched the mir-like gene precursor sequence located at ch5 positions 13644905–1364500. Figure 10N shows the secondary structure of the mir-like precursor ath_dead_mir1334. [Figure 11A-D]Figures 11A–J provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir247 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-8180 (MI0026810). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. We used the RNAplot module in the ViennaRNA package to generate secondary structures for each precursor sequence. Specifically, Figure 11E shows the secondary structure of the WT miRNA precursor described above. Figure 11F shows a distribution plot for all 21-bp-long small RNA-seq reads from roots that perfectly matched the miRNA-like gene precursor sequence. Figure 11J shows the secondary structure of the mir-like precursor ath_dead_mir247. [Figure 11E-H]Figures 11A–J provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir247 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-8180 (MI0026810). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. We used the RNAplot module in the ViennaRNA package to generate secondary structures for each precursor sequence. Specifically, Figure 11E shows the secondary structure of the WT miRNA precursor described above. Figure 11F shows a distribution plot for all 21-bp-long small RNA-seq reads from roots that perfectly matched the miRNA-like gene precursor sequence. Figure 11J shows the secondary structure of the mir-like precursor ath_dead_mir247. [Figure 11I-J]Figures 11A–J provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir247 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-8180 (MI0026810). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. We used the RNAplot module in the ViennaRNA package to generate secondary structures for each precursor sequence. Specifically, Figure 11E shows the secondary structure of the WT miRNA precursor described above. Figure 11F shows a distribution plot for all 21-bp-long small RNA-seq reads from roots that perfectly matched the miRNA-like gene precursor sequence. Figure 11J shows the secondary structure of the mir-like precursor ath_dead_mir247. [Figure 12A-D]Figures 12A-I provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir859 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-405a (MI0001074). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 12A shows a distribution plot for all 24-bp-long root small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene ath-mir-405a). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 12D shows the secondary structure of the previously described WT miRNA precursor. Figure 12E shows a distribution plot of all 23-bp-long root smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 12I shows the secondary structure of the mir-like precursor ath_dead_mir859. [Figure 12E-H]Figures 12A-I provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir859 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-405a (MI0001074). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 12A shows a distribution plot for all 24-bp-long root small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene ath-mir-405a). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 12D shows the secondary structure of the previously described WT miRNA precursor. Figure 12E shows a distribution plot of all 23-bp-long root smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 12I shows the secondary structure of the mir-like precursor ath_dead_mir859. [Figure 12I]Figures 12A-I provide plots of the small RNA distribution and secondary structure of the miRNA-like gene ath_dead_mir859 from Arabidopsis thaliana and its corresponding WT miRNA ath-mir-405a (MI0001074). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, and a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were created if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 12A shows a distribution plot for all 24-bp-long root small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene ath-mir-405a). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 12D shows the secondary structure of the previously described WT miRNA precursor. Figure 12E shows a distribution plot of all 23-bp-long root smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 12I shows the secondary structure of the mir-like precursor ath_dead_mir859. [Figure 13A-D]Figures 13A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir219 from C. elegans and its corresponding WT miRNA cel-mir-5545 (MI0019066). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 13A shows a distribution plot for all 21-bp-long small RNA-seq reads from embryos that perfectly matched the precursor sequence of the WT miRNA gene cell-mir-5545. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 13B shows a distribution plot of all 22-bp-long embryonic small RNA-seq reads that perfectly matched the WT precursor sequence. Figure 13E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 13F shows a distribution plot of all 22-bp-long young adult small RNA-seq reads that perfectly matched the mir-like gene precursor sequence. Figure 13H shows the secondary structure of the mir-like precursor cel_dead_mir219. [Figure 13E-H]Figures 13A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir219 from C. elegans and its corresponding WT miRNA cel-mir-5545 (MI0019066). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 13A shows a distribution plot for all 21-bp-long small RNA-seq reads from embryos that perfectly matched the precursor sequence of the WT miRNA gene cell-mir-5545. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 13B shows a distribution plot of all 22-bp-long embryonic small RNA-seq reads that perfectly matched the WT precursor sequence. Figure 13E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 13F shows a distribution plot of all 22-bp-long young adult small RNA-seq reads that perfectly matched the mir-like gene precursor sequence. Figure 13H shows the secondary structure of the mir-like precursor cel_dead_mir219. [Figure 14A-D]Figures 14A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir363 from C. elegans and its corresponding WT miRNA cel-mir-5545 (MI0019066). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 14A shows a distribution plot for all 21-bp-long small RNA-seq reads from embryos that perfectly matched the precursor sequence of the WT miRNA gene cel-mir-5545. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 14B shows a distribution plot for all 22-bp-long embryonic smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 14E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 14F shows a distribution plot for all L4 22-bp-long smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 14H shows the secondary structure of the mir-like precursor cel_dead_mir363. [Figure 14E-H]Figures 14A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir363 from C. elegans and its corresponding WT miRNA cel-mir-5545 (MI0019066). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 14A shows a distribution plot for all 21-bp-long small RNA-seq reads from embryos that perfectly matched the precursor sequence of the WT miRNA gene cel-mir-5545. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 14B shows a distribution plot for all 22-bp-long embryonic smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 14E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 14F shows a distribution plot for all L4 22-bp-long smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 14H shows the secondary structure of the mir-like precursor cel_dead_mir363. [Figure 15A-D]Figures 15A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir537 from C. elegans and its corresponding WT miRNA cel-mir-8196b (MI0026837). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 15A shows a distribution plot of all 23-bp-long embryonic small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene cel-mir-8196b). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 15F shows the secondary structure of the WT miRNA precursor described above. Figure 15G shows a distribution plot of all embryonic smallRNA-seq reads that perfectly matched the mir-like gene precursor sequence. Figure 15H shows the secondary structure of the mir-like precursor cel_dead_mir537. Note that the WT and mir-like sequences differ by only a few bases. Therefore, their secondary structures are predicted to be very similar or even identical. [Figure 15E-F]Figures 15A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir537 from C. elegans and its corresponding WT miRNA cel-mir-8196b (MI0026837). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 15A shows a distribution plot of all 23-bp-long embryonic small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene cel-mir-8196b). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 15F shows the secondary structure of the WT miRNA precursor described above. Figure 15G shows a distribution plot of all embryonic smallRNA-seq reads that perfectly matched the mir-like gene precursor sequence. Figure 15H shows the secondary structure of the mir-like precursor cel_dead_mir537. Note that the WT and mir-like sequences differ by only a few bases. Therefore, their secondary structures are predicted to be very similar or even identical. [Figure 15G-H]Figures 15A–H provide plots of the small RNA distribution and secondary structure of the miRNA-like gene cel_dead_mir537 from C. elegans and its corresponding WT miRNA cel-mir-8196b (MI0026837). For each mir-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group named "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 15A shows a distribution plot of all 23-bp-long embryonic small RNA-seq reads that perfectly matched the WT precursor sequence (miRNA gene cel-mir-8196b). The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Figure 15F shows the secondary structure of the WT miRNA precursor described above. Figure 15G shows a distribution plot of all embryonic smallRNA-seq reads that perfectly matched the mir-like gene precursor sequence. Figure 15H shows the secondary structure of the mir-like precursor cel_dead_mir537. Note that the WT and mir-like sequences differ by only a few bases. Therefore, their secondary structures are predicted to be very similar or even identical. [Figures 16A-D]Figures 16A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54024 and its corresponding WT miRNA hsa-mir-523 (MI0003153). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 16A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-523. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 16B shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 16E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 16I shows a distribution plot for all lung smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 16F shows the secondary structure of the mir-like precursor hsa_dead_mir54024. [Figure 16E-H]Figures 16A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54024 and its corresponding WT miRNA hsa-mir-523 (MI0003153). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 16A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-523. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 16B shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 16E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 16I shows a distribution plot for all lung smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 16F shows the secondary structure of the mir-like precursor hsa_dead_mir54024. [Figure 16I-J]Figures 16A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54024 and its corresponding WT miRNA hsa-mir-523 (MI0003153). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 16A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-523. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 16B shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 16E shows the secondary structure of the aforementioned WT miRNA precursor. Figure 16I shows a distribution plot for all lung smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 16F shows the secondary structure of the mir-like precursor hsa_dead_mir54024. [Figures 17A-D]Figures 17A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54573 and its corresponding WT miRNA hsa-mir-663b (MI0006336). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 17A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-663b. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 17B shows a distribution plot for all brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 17C shows the secondary structure of the WT miRNA precursor hsa-mir-663b. Figure 17D shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 17J shows the secondary structure of the mir-like precursor hsa_dead_mir54573. [Figure 17E-H]Figures 17A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54573 and its corresponding WT miRNA hsa-mir-663b (MI0006336). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 17A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-663b. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 17B shows a distribution plot for all brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 17C shows the secondary structure of the WT miRNA precursor hsa-mir-663b. Figure 17D shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 17J shows the secondary structure of the mir-like precursor hsa_dead_mir54573. [Figure 17I-J]Figures 17A–J provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir54573 and its corresponding WT miRNA hsa-mir-663b (MI0006336). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 17A shows the distribution plot for all 21-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-663b. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 17B shows a distribution plot for all brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 17C shows the secondary structure of the WT miRNA precursor hsa-mir-663b. Figure 17D shows a distribution plot for all 22-bp-long brain smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 17J shows the secondary structure of the mir-like precursor hsa_dead_mir54573. [Figures 18A-C]Figures 18A–E provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir50078 and its corresponding WT miRNA hsa-mir-1273h (MI0025512). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 18A shows the distribution plot for all 23-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-1273h. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 18B shows a distribution plot for all brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 18C shows the secondary structure of the aforementioned WT miRNA precursor. Figure 18D shows a distribution plot for all brain smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 18E shows the secondary structure of the mir-like precursor hsa_dead_mir50078. [Figure 18D-E]Figures 18A–E provide plots of the small RNA distribution and secondary structure of the human miRNA-like gene hsa_dead_mir50078 and its corresponding WT miRNA hsa-mir-1273h (MI0025512). For each miRNA-like gene and its corresponding WT miRNA, we plotted the distribution of reads that perfectly matched the corresponding precursor sequence using seven different read size groups ranging from 19 to 24 bp in length, as well as a group designated "small molecules" representing small RNA-seq reads of all sizes. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate the secondary structure of each precursor sequence. Specifically, Figure 18A shows the distribution plot for all 23-bp-long brain small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene hsa-mir-1273h. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 18B shows a distribution plot for all brain smallRNAseq reads that perfectly matched the WT precursor sequence. Figure 18C shows the secondary structure of the aforementioned WT miRNA precursor. Figure 18D shows a distribution plot for all brain smallRNAseq reads that perfectly matched the mir-like gene precursor sequence. Figure 18E shows the secondary structure of the mir-like precursor hsa_dead_mir50078. [Figure 19A-D]Figures 19A–H provide plots of the small RNA distribution and secondary structure of the C. elegans miRNA cel-mir-71 (MI0000042). The distribution of reads that perfectly matched the mRNA precursor sequence was plotted using seven different read size groups ranging from 19 to 24 bp in length, and a group designated "small molecules" representing all sizes of small RNA-seq reads. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate secondary structure plots for each precursor sequence. Specifically, Figure 19A shows the distribution plot for all 21-bp-long embryonic small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene cel-mir-71. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 19B shows a distribution plot of all 23-bp-long embryonic smallRNAseq reads that perfectly matched the precursor sequence. Figure 19H shows the secondary structure of miRNAcel-mir-71. [Figure 19E-F]Figures 19A–H provide plots of the small RNA distribution and secondary structure of the C. elegans miRNA cel-mir-71 (MI0000042). The distribution of reads that perfectly matched the mRNA precursor sequence was plotted using seven different read size groups ranging from 19 to 24 bp in length, and a group designated "small molecules" representing all sizes of small RNA-seq reads. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate secondary structure plots for each precursor sequence. Specifically, Figure 19A shows the distribution plot for all 21-bp-long embryonic small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene cel-mir-71. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 19B shows a distribution plot of all 23-bp-long embryonic smallRNAseq reads that perfectly matched the precursor sequence. Figure 19H shows the secondary structure of miRNAcel-mir-71. [Figure 19G-H]Figures 19A–H provide plots of the small RNA distribution and secondary structure of the C. elegans miRNA cel-mir-71 (MI0000042). The distribution of reads that perfectly matched the mRNA precursor sequence was plotted using seven different read size groups ranging from 19 to 24 bp in length, and a group designated "small molecules" representing all sizes of small RNA-seq reads. Read counts were normalized to RPKM, and plots for specific size groups were generated if there were at least 10 reads that perfectly matched the corresponding precursor sequence. The RNAplot module of the ViennaRNA package was used to generate secondary structure plots for each precursor sequence. Specifically, Figure 19A shows the distribution plot for all 21-bp-long embryonic small RNA-seq reads that perfectly matched the precursor sequence of the WT miRNA gene cel-mir-71. The bar graph below each plot marks the position of the mature sequence of the plotted precursor, and the legend indicates the size of the mature sequence. Similarly, Figure 19B shows a distribution plot of all 23-bp-long embryonic smallRNAseq reads that perfectly matched the precursor sequence. Figure 19H shows the secondary structure of miRNAcel-mir-71. DETAILED DESCRIPTION OF THE INVENTION

[0097] In some embodiments, the present invention relates to conferring silencing activity to silencing-dysfunctional RNA molecules (e.g., miRNA-like molecules) in eukaryotic cells, and in some cases altering the silencing specificity of the RNA molecules for silencing an endogenous or exogenous target RNA of interest.

[0098] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0099] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following specification or illustrated by way of examples. The invention is capable of other embodiments or of being practiced or carried out in various ways and in different organisms. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0100] Previous studies on genome editing of RNA molecules in various organisms (e.g., mice, humans, and plants) have focused on disrupting miRNA activity or target binding sites using gene transfer. Genome editing in plants has focused on using nucleases such as CRISPR-Cas9 technology, ZFN, and TALEN to knock down genes or insertions in model plants. Furthermore, gene silencing in plants has also been reported using artificial miRNA transgenes to silence endogenous and exogenous target genes [Molnar A et al. Plant J. (2009)]. )58(1):165-74.Doi:10.1111 / j.1365-313X.2008.03767.x.Epub 2009 Jan 19;Borges and Martienssen,Nature Reviews Molecular Cell Biology | AOP, published online 4 November 2015; doi:10.1038 / nrm4085]. Artificial miRNA transgenes are introduced into plant cells within artificial expression cassettes (containing promoters, terminators, selectable markers, etc.) to downregulate the expression of their targets.

[0101] Gene therapy techniques developed in mammalian organisms (e.g., for human treatment) include gene therapy, which can restore lost gene function through the expression of viral transgenes, and RNAi, which mediates the suppression of defective genes by knocking down target mRNA. Recent advances in genome editing technology have also made it possible to alter the DNA sequence of living cells by inducing site-specific double-strand breaks (DSBs) at desired locations in the genome and then editing one or more nucleotides in the cells of human patients using genome editing techniques (e.g., NHEJ and HR). While NHEJ is primarily, but not exclusively, used for knockout purposes, HR is used to introduce precise edits at specific sites, such as point mutations, or to correct naturally occurring or inherited deleterious mutations.

[0102] The present invention is based, in part, on the identification of genes encoding RNA molecules, where (1) the RNA molecules encoded by the identified genes exhibit homology to corresponding canonical silencing RNA molecules (e.g., miRNAs and / or miRNA precursors) from the same organism; (2) the identified genes are transcribed into RNA molecules; and (3) the RNA expressed by the identified genes is not processed into RNA in the same way as the corresponding homologous canonical silencing molecules (i.e., the RNA expressed by the identified genes is aberrantly processed or not processed). As exemplified herein below, such genes have been identified in a variety of organisms. Without being bound by theory or mechanism, the identified genes encode RNA molecules that are dysfunctional in silencing because such aberrantly processed RNAs are not processed into RNA molecules with silencing activity.

[0103] In putting the invention into practical use, the inventors have devised a gene editing technique aimed at conferring standard processibility (e.g., processing by an RNAi factor such as Dicer) on a dysfunctional RNA molecule, wherein the dysfunctional RNA molecule contains at least one nucleic acid sequence change relative to a homologous nucleic acid sequence encoding a standardly processed RNA molecule in the same organism, and further wherein the dysfunctional RNA molecule is transcribed intracellularly.

[0104] The inventors have further utilized gene editing techniques to redirect the silencing specificity of processible RNA molecules to target and interfere with the expression of a target gene of interest (endogenous or exogenous to the cell) that was not originally targeted by the silencing RNA. Specifically, the inventors have designed a genome editing-induced gene silencing (GEiGS) platform that utilizes endogenous RNA molecules of eukaryotic cells, including non-coding RNA molecules (e.g., RNA silencing molecules, e.g., siRNA, miRNA, piRNA, tasiRNA, tRNA, rRNA, antisense RNA, etc.), and can modify them to target any RNA target of interest. In this method, GEiGS is used to edit a few nucleotides in these endogenous RNA molecules, thereby redirecting their activity and / or specificity to effectively and specifically target any RNA of interest. The gene editing techniques described herein can be achieved using standard molecular genetic tools and transfection systems, including expression cassettes with promoters, terminators, and selectable markers. Furthermore, the gene editing techniques of some embodiments of the present invention involve genome editing of RNA molecules (e.g., endogenous) that are stable and heritable.

[0105] Thus, according to one aspect of the present invention, there is provided a method for generating RNA molecules having silencing activity in a cell, comprising: (a) identifying nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology, not including complete identity, to nucleic acid sequences encoding RNA molecules that associate with an RNA-induced silencing complex (RISC); (b) determining the transcription of the nucleic acid sequences encoding the RNA molecules to select transcribable nucleic acid sequences encoding the RNA molecules that exhibit the predetermined range of sequence homology; and (c) determining the transcription of the nucleic acid sequences encoding the transcribable nucleic acid sequences encoding the RNA molecules that exhibit the predetermined range of sequence homology. The method includes (a) determining the processivity into small RNAs of transcripts of a transcribable nucleic acid sequence encoding an RNA molecule exhibiting the predetermined sequence homology range to select a nucleic acid sequence, wherein the RNA molecule is aberrantly processed; and (d) modifying the nucleic acid sequence of the transcribable nucleic acid sequence encoding the aberrantly processed RNA molecule exhibiting the predetermined sequence homology range to confer processivity into small RNAs that associate with RISC and are complementary to a first target RNA, thereby generating an RNA molecule with silencing activity in a cell.

[0106] According to some embodiments, there is provided a method for generating RNA molecules having silencing activity in a cell, comprising: (a) selecting nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology, not including complete identity, to a nucleic acid sequence encoding an RNA molecule that associates with an RNA-induced silencing complex (RISC), wherein the selection comprises: (1) determining transcription of the nucleic acid sequences encoding the RNA molecules to select transcribable nucleic acid sequences encoding the RNA molecules that exhibit the predetermined range of sequence homology; and (2) determining transcription of the transcribable nucleic acid sequences encoding the RNA molecules that exhibit the predetermined range of sequence homology. Provided herein are methods comprising: (a) determining the processivity into small RNAs of transcripts of transcribable nucleic acid sequences encoding RNA molecules exhibiting the predetermined sequence homology range to select a sequence, wherein the RNA molecule is aberrantly processed; and (b) modifying the nucleic acid sequence of the transcribable nucleic acid sequence encoding the aberrantly processed RNA molecule exhibiting the predetermined sequence homology range to confer processivity into small RNAs that associate with RISC and are complementary to a first target RNA, thereby generating an RNA molecule with silencing activity in a cell.

[0107] According to one embodiment, the cell is a eukaryotic cell.

[0108] The term "eukaryotic cell," as used herein, refers to any cell of a eukaryotic organism. Eukaryotes include unicellular and multicellular organisms. Unicellular eukaryotes include, but are not limited to, yeast, protists, slime molds, and algae. Multicellular eukaryotes include, but are not limited to, animals (e.g., mammals, insects, invertebrates, nematodes, birds, fish, reptiles, and crustaceans), plants, fungi, and algae (e.g., brown algae, red algae, blue-green algae).

[0109] According to one embodiment, the cell is a plant cell.

[0110] According to a particular embodiment, the plant cell is a protoplast.

[0111] Protoplasts may be derived from any plant tissue, such as fruit, flowers, roots, leaves, embryos, embryonic cell suspensions, callus, or seedling tissue (as described below).

[0112] According to certain embodiments, the plant cell is an embryogenic cell.

[0113] According to certain embodiments, the plant cells are somatic embryogenic cells.

[0114] According to one embodiment, the eukaryotic cell is not a plant cell.

[0115] According to one embodiment, the eukaryotic cell is an animal cell (eg, a non-human animal cell).

[0116] According to one embodiment, the eukaryotic cell is a vertebrate cell.

[0117] According to one embodiment, the eukaryotic cell is an invertebrate cell.

[0118] According to certain embodiments, the invertebrate cells are cells of insects, snails, clams, octopuses, starfish, sea urchins, jellyfish, and caterpillars.

[0119] According to certain embodiments, the invertebrate cells are crustacean cells. Exemplary crustaceans include, but are not limited to, shrimp, prawns, crabs, lobsters, and crayfish.

[0120] According to certain embodiments, the invertebrate cells are fish cells. Exemplary fish include, but are not limited to, salmon, tuna, pollock, catfish, cod, haddock, prawn, sea bass, tilapia, Arctic char, and carp.

[0121] According to one embodiment, the eukaryotic cell is a mammalian cell (eg, a non-human mammalian cell).

[0122] According to certain embodiments, the mammalian cells are cells of non-human organisms such as, but not limited to, rodents, rabbits, pigs, goats, ruminants (e.g., cows, sheep, antelopes, deer, and giraffes), dogs, cats, horses, and non-human primates.

[0123] According to a particular embodiment, the eukaryotic cell is a human cell.

[0124] According to one embodiment, the eukaryotic cell is a primary cell, a cell line, a somatic cell, a germ cell, a stem cell, an embryonic stem cell, an adult stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell (iPS), a gamete cell, a zygote cell, a blastocyst cell, an embryo, a fetus, and / or a donor cell.

[0125] As used herein, the phrase "stem cell" refers to a cell (e.g., a totipotent, pluripotent, or multipotent stem cell) that can remain in an undifferentiated state in culture for extended periods of time until induced to differentiate into another cell type (e.g., a fully differentiated cell) with a specific specialized function. Totipotent cells, such as embryonic cells that have undergone only the first few cell divisions after fertilization, can differentiate into embryonic and extraembryonic cells and are the only cells that can develop into a viable human being. Preferably, the phrase "pluripotent stem cell" refers to a cell that can differentiate into all three embryonic germ layers, i.e., ectoderm, endoderm, and mesoderm, or remain in an undifferentiated state. Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS). Multipotent stem cells include adult stem cells and hematopoietic stem cells.

[0126] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., ectoderm, endoderm, and mesoderm) or can remain undifferentiated. The term "embryonic stem cells" includes cells obtained from embryonic tissue formed after conception (e.g., blastocysts), before implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (WO 2006 / 040763). ), embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before 10 weeks of gestation, and cells originating from unfertilized eggs stimulated by parthenogenesis (parthenogenetic organisms).

[0127] Embryonic stem cells in some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage.

[0128] It will be appreciated that commercially available stem cells may be used in accordance with some embodiments of the present invention. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry [www(dot)grants(dot)nih(dot)gov / stem_cells / registry / current(dot)html].

[0129] Embryonic stem cells have also been used to generate new stem cells in mice (Mills and Bradley, 2001), golden hamsters (Doetschman et al., 1988, Dev Biol. 127:224-7), rats (Iannaccone et al., 1994, Dev Biol. 163:288-92), rabbits (Giles et al. 1993, Mol Reprod Dev. 36:130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36:424-33), and several livestock species (Notarianni et al., 1991, J Reprod Fertil Suppl. 43:255-60; Wheeler 1994, Reprod Fertil Dev. 6:563-8; Mitalipova et al., 2001, Cloning. 3:59-67], and non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55:254-9].

[0130] "Induced pluripotent stem cells" (iPS; embryonic-like stem cells) refer to cells obtained by dedifferentiating adult somatic cells that have been rendered pluripotent (i.e., capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm, and mesoderm). According to some embodiments of the present invention, such cells are obtained from differentiated tissues (e.g., somatic tissues such as skin) and undergo dedifferentiation by genetic manipulation that reprograms the cells to acquire embryonic stem cell characteristics. According to some embodiments of the present invention, induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4, and c-Myc in somatic stem cells.

[0131] Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can be generated from somatic cells, such as fibroblasts, hepatocytes, and gastric epithelial cells, by genetic manipulation of the somatic cells, for example, by retroviral transduction of transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 [see, e.g., Park et al. Reprogramming of somatic cells]. Human somatic cells to pluripotency with defined factors. Nature (2008) 451:141-146].

[0132] The phrase "adult stem cells" (also called "tissue stem cells" or stem cells derived from somatic tissue) refers to any stem cell derived from somatic tissue [of a postnatal or prenatal animal, particularly a human]. Adult stem cells are generally considered to be multipotent stem cells, capable of differentiating into multiple cell types. Adult stem cells are found in adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow, and The tissue may be derived from any adult, neonatal, or fetal tissue, including placenta.

[0133] According to one embodiment, the stem cells utilized by some embodiments of the present invention are bone marrow (BM) derived stem cells, including hematopoietic stem cells, stromal stem cells, or mesenchymal stem cells [Dominici, M et al., (2001) J. Biol. Regul. Homeost. Agents. 15:28-37]. BM derived stem cells can be obtained from the iliac crest, femur, tibia, spine, rib, or other medullary cavity.

[0134] Hematopoietic stem cells (HSCs), sometimes referred to as adult tissue stem cells, include stem cells obtained from the blood or bone marrow tissue of individuals of any age, or from the umbilical cord blood of newborn individuals. Preferred stem cells according to this aspect of some embodiments of the invention are embryonic stem cells, preferably of human or primate (e.g., monkey) origin.

[0135] Placental and umbilical cord blood stem cells are sometimes referred to as "juvenile stem cells."

[0136] Mesenchymal stem cells (MSCs), which are formative pluripotent blast cells, give rise to one or more mesenchymal tissues (e.g., fat, bone, cartilage, elastic and fibrous connective tissue, myoblasts) and tissues other than those of embryonic mesodermal origin (e.g., neural cells), depending on various influences from bioactive factors such as cytokines. Although such cells can be isolated from embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood, and other tissues, their isolation from BM is currently preferred because their abundance in BM far exceeds their abundance in other tissues.

[0137] Adult tissue stem cells can be isolated using various methods known in the art, such as those disclosed by Alison, MR [J Pathol. (2003) 200(5):547-50]. Fetal stem cells can be isolated using various methods known in the art, such as those disclosed by Eventov-Friedman S et al. [PloS Med. (2006) 3:e215].

[0138] Hematopoietic stem cells can be isolated using various methods known in the art, such as those disclosed in "Handbook of Stem Cells", edited by Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp. 609-614, "Isolation and Characterization of Hematopoietic Stem Cells", by Gerald J Spangrude and William B Stayton.

[0139] Methods for isolating, purifying, and expanding mesenchymal stem cells (MSCs) are known in the art, including those disclosed, for example, by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and by Jones EA et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12):3349-60.

[0140] According to one embodiment, the eukaryotic cell is isolated from its natural environment (eg, the human body).

[0141] According to one embodiment, the eukaryotic cell is a healthy cell.

[0142] According to one embodiment, the eukaryotic cell is a diseased cell or a cell susceptible to a disease.

[0143] According to one embodiment, the eukaryotic cell is a cancer cell.

[0144] According to one embodiment, the eukaryotic cell is an immune cell (eg, a T cell, a B cell, a macrophage, a NK cell, etc.).

[0145] According to one embodiment, the eukaryotic cell is a cell infected with a pathogen (eg, a bacterial, viral, or fungal pathogen).

[0146] The term "RNA molecule with silencing activity" or "RNA silencing molecule" refers to a non-coding RNA (ncRNA) molecule, i.e., an RNA sequence that is not translated into an amino acid sequence, does not code for a protein, and is capable of mediating RNA silencing or interference (RNAi).

[0147] The term "RNA silencing" or "RNAi" refers to a cellular regulatory mechanism in which non-coding RNA molecules ("RNA molecules with silencing activity" or "RNA silencing molecules") mediate the co- or post-transcriptional inhibition of gene expression or translation in a sequence-specific manner.

[0148] According to one embodiment, the RNA silencing molecule is capable of mediating the suppression of RNA during transcription (co-transcriptional gene silencing).

[0149] According to certain embodiments, co-transcriptional gene silencing comprises epigenetic silencing (e.g., a chromosomal condition that prevents functional gene expression).

[0150] According to one embodiment, the RNA silencing molecule is capable of mediating RNA suppression after transcription (post-transcriptional gene silencing).

[0151] Post-transcriptional gene silencing (PTGS) typically refers to the process (typically occurring in the cytoplasm of a cell) of degradation or cleavage of messenger RNA (mRNA) molecules, which reduces their activity by preventing translation. For example, as described in detail below, the guide strand of an RNA silencing molecule pairs with a complementary sequence in an mRNA molecule and induces cleavage, e.g., by Argonaute 2 (Ago2). Specifically, members of the Argonaute (Ago) protein family function as direct interaction partners of RNA silencing molecules within the RNA-induced silencing complex (RISC). The RNA silencing molecule serves to guide RISC to its target mRNA, while the Ago protein complex suppresses mRNA translation or induces deadenylation-dependent mRNA decay, resulting in silencing of gene expression.

[0152] Co-transcriptional gene silencing typically refers to the inactivation of gene activity (i.e., transcriptional repression) and typically occurs in the cell nucleus. This repression of gene activity is mediated by epigenetic factors, such as methyltransferases, which methylate target DNA and histones. Thus, in co-transcriptional gene silencing, binding of small RNAs to target RNAs (small RNA-transcript interactions) destabilizes the target nascent transcript and recruits enzymes that modify DNA and histones (i.e., epigenetic factors), which induce chromatin remodeling into structures that repress gene activity and transcription. Furthermore, in co-transcriptional gene silencing, long non-coding RNA scaffolds bound to chromatin can recruit chromatin-modifying complexes independently of small RNAs. These co-transcriptional silencing mechanisms form an RNA surveillance system that detects and silences inappropriate transcriptional events and provides memory of these events through a self-reinforcing epigenetic loop [D. Hoch and D. Moazed, RNA-mediated epigenetic repression, 2009]. genetic regulation of gene expression, as described in Nat Rev Genet. (2015) 16(2):71-84].

[0153] Below is a detailed description of RNA silencing molecules that associate with an RNA-induced silencing complex (RISC) and have intrinsic RNAi activity (e.g., are RNA silencing molecules) that can be used in accordance with certain embodiments of the present invention.

[0154] Perfectly and imperfectly paired RNAs (i.e., double-stranded RNAs; dsRNAs), siRNAs, and shRNAs—the presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer (also known as endoribonuclease Dicer or helicase with an RNase motif) is an enzyme typically referred to as a Dicer-like (DCL) protein in plants. The number of DCL genes varies among plants; for example, the Arabidopsis genome typically has four DCL genes, rice has eight DCL genes, and the maize genome has five DCL genes. Dicer is involved in processing dsRNA into short pieces of dsRNA known as small interfering RNAs (siRNAs). siRNAs derived from Dicer activity are typically about 21 to about 23 nucleotides long and contain approximately 19 base pair duplexes with two 3' nucleotide overhangs.

[0155] According to one embodiment, dsRNA precursors longer than 21 bp are used. Various studies have demonstrated that long dsRNAs can be used to silence gene expression without inducing stress responses or causing significant off-target effects—for example, [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al.Brain Res.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].

[0156] The term "siRNA" refers to small inhibitory RNA duplexes (typically 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21-mers with a central 19-bp duplex region and symmetric two-base 3' overhangs at the termini. However, it has recently been reported that chemically synthesized RNA duplexes of 25-30 bases in length can exhibit up to 100-fold increased potency compared to 21-mers at the same positions. The observed increased potency obtained with longer RNAs in inducing RNAi is due to providing Dicer with a substrate (27-mer) rather than a product (21-mer), suggesting that this improves the speed or efficiency of RISC entry of siRNA duplexes.

[0157] The position, but not the composition, of the 3' overhang influences the potency of siRNA, and asymmetric duplexes with a 3' overhang on the antisense strand have generally been found to be more potent than those with a 3' overhang on the sense strand (Rose et al., 2005).

[0158] The strands of double-stranded interfering RNA (e.g., siRNA) can be joined to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as mentioned above, the RNA silencing molecule of some embodiments of the present invention can be a short hairpin RNA (shRNA).

[0159] The term "shRNA," as used herein, refers to a short hairpin RNA molecule having a stem-loop structure, comprising first and second regions of complementary sequence, the degree and orientation of which are sufficient to allow base pairing between the regions, the first and second regions joined by a loop region, and the loop resulting from the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is between 3 and 23, or between 5 and 15, or between 7 and 13, or between 4 and 9, or between 9 and 11. Some of the nucleotides in the loop may participate in base-pairing interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form a loop include 5'-CAAGAGA-3' and 5'-UUACAA-3' (WO2013126963 and WO2014107763). Those skilled in the art will recognize that the resulting single-stranded oligonucleotide forms a stem-loop or hairpin structure that contains a double-stranded region that can interact with the RNAi machinery.

[0160] The RNA silencing molecules of some embodiments of the present invention need not be limited to molecules containing only RNA, but also encompass chemically modified nucleotides and non-nucleotides.

[0161] Various types of siRNAs are contemplated by the present invention, including trans-acting siRNAs (Ta-siRNAs or TasiRNAs), repeat-associated siRNAs (Ra-siRNAs), and siRNAs derived from natural antisense transcripts (Nat-siRNAs).

[0162] According to one embodiment, the silencing RNA comprises "piRNA," a class of Piwi-binding RNAs approximately 26 and 31 nucleotides in length. piRNAs typically form RNA-protein complexes through interactions with Piwi proteins, i.e., antisense piRNAs are typically loaded onto Piwi proteins (e.g., Piwi, Ago3, and Aubergine (Aub)).

[0163] miRNA - According to another embodiment, the RNA silencing molecule may be a miRNA.

[0164] The terms "microRNA," "miRNA," and "miR" are synonymous and refer to a collection of non-coding, single-stranded RNA molecules approximately 19-24 nucleotides in length that regulate gene expression. miRNAs are found in a wide range of organisms (e.g., insects, mammals, plants, and nematodes) and have been shown to play roles in development, homeostasis, and disease pathogenesis.

[0165] Pre-miRNAs initially exist as long, imperfect double-stranded stem-loop RNAs that are synthesized by Dicer into mature guide strands (miRNAs) and similarly sized fragments known as passenger strands (miRNAs). * ) are further processed into siRNA-like duplexes containing miRNA and miRNA * can be derived from opposing arms of the pri-miRNA and pre-miRNA. * The sequences may also be found in libraries of cloned miRNAs, but typically at lower frequencies than miRNAs.

[0166] First, miRNA * Although miRNAs exist as double-stranded species along with miRNAs, they are ultimately incorporated as single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). A variety of proteins can form RISC, which allows the miRNA / miRNA complex to be integrated into the ribonucleoprotein complex. * Duplex specificity, binding site of target gene, miRNA activity (repression or activation), and miRNA / miRNA * Which strand of the duplex is loaded into RISC can vary.

[0167] miRNA:miRNA * When the double-stranded miRNA strand is loaded into RISC, the miRNA *miRNA is removed and degraded. miRNA loaded into RISC: miRNA * The strand of the duplex is the one that is less tightly paired at the 5' end. miRNA:miRNA * If both ends of the miRNA have approximately equivalent 5' pairing, the miRNA and miRNA * Both may have gene silencing activity.

[0168] RISC identifies target nucleic acids based on the high level of complementarity between miRNA and mRNA, specifically by the second to eighth nucleotides of the miRNA (referred to as the "seed sequence").

[0169] Numerous studies have investigated the base-pairing requirements between miRNAs and their mRNA targets for efficient translation inhibition (reviewed by Bartel 2004, Cell 116-281). Computational studies analyzing genome-wide miRNA binding suggest that bases 2-8 of the 5' end of the miRNA (also known as the "seed sequence") play a special role in target binding, although the role of the first nucleotide, usually found to be "A," has also been recognized (Lewis et al. 2005, Cell 120-15). Similarly, Krek et al. (2005, Nat Genet 37-495) identified and validated targets using nucleotides 1-7 or 2-8. Target sites in mRNAs can be located in the 5' UTR, 3' UTR, or coding region. Interestingly, multiple miRNAs can regulate the same mRNA target by recognizing the same site or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs results in the most efficient translation inhibition.

[0170] miRNAs can instruct RISC to downregulate gene expression through one of two mechanisms: mRNA cleavage or translational repression. If the mRNA has a certain degree of complementarity to the miRNA, the miRNA can direct mRNA cleavage. When the miRNA induces cleavage, cleavage typically occurs between the nucleotide pairings with the 10th and 11th residues of the miRNA. Alternatively, if the miRNA does not have the required degree of complementarity to the miRNA, the miRNA can repress translation. Translational repression may be more prevalent in animals, as the degree of complementarity between the miRNA and the binding site may be lower in animals.

[0171] miRNA and miRNA * It should be noted that there may be variability in the 5' and 3' ends of any pair of miRNAs. This variability may be due to variability in the enzymatic processing of Drosha and Dicer relative to the cleavage site. * The variation in the 5' and 3' ends of the miRNAs may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. Mismatches in the stem strands may lead to a population of different hairpin structures. Variation in stem structure may also lead to variations in the products of Drosha and Dicer cleavage.

[0172] According to one embodiment, miRNAs can be processed independently of Dicer, for example, by Argonaute 2.

[0173] It will be understood that a pre-miRNA sequence may comprise 45 to 90, 60 to 80, or 60 to 70 nucleotides, while a pri-miRNA sequence may comprise 45 to 30,000, 50 to 25,000, 100 to 20,000, 1,000 to 1,500, or 80 to 100 nucleotides.

[0174] Antisense - Antisense is a single-stranded RNA designed to specifically hybridize with the mRNA of a gene, thereby blocking or inhibiting the expression of that gene. Antisense polynucleotides capable of specifically hybridizing with the mRNA transcript encoding the target RNA can be used to downregulate the target RNA.

[0175] transposable element RNA Transposable elements (Te) contain vast DNA sequences, all of which have the ability to move to new sites in the genome either directly (transposons) by a cut-and-paste mechanism or indirectly via an RNA intermediate (retrotransposons). Te are divided into autonomous and non-autonomous classes depending on whether they contain ORFs encoding proteins required for transposition. RNA-mediated gene silencing is one of the mechanisms by which the genome controls Te activity and the deleterious effects resulting from genetic and epigenetic instability of the genome.

[0176] In one embodiment, the RNA silencing molecule may associate with RISC but may not have standard (intrinsic) RNAi activity (e.g., it is not a standard RNA silencing molecule or its target has not been identified). Such RNA silencing molecules include:

[0177] According to one embodiment, the RNA silencing molecule is a transfer RNA (tRNA) or a transfer RNA fragment (tRF). The term "tRNA" refers to an RNA molecule that serves to physically link the nucleotide sequence of a nucleic acid with the amino acid sequence of a protein, and was previously referred to as soluble RNA or sRNA. tRNAs are typically about 76-90 nucleotides in length. According to one embodiment, the RNA silencing molecule is a ribosomal RNA (rRNA). The term "rRNA" refers to the RNA component of the ribosome, i.e., either the small ribosomal subunit or the large ribosomal subunit.

[0178] In one embodiment, the RNA silencing molecule is a small nuclear RNA (snRNA or U-RNA). The term "sRNA" or "U-RNA" refers to a small RNA molecule found in the splicing speckles and Cajal bodies of the nucleus of eukaryotic cells. snRNAs are typically about 150 nucleotides in length.

[0179] According to one embodiment, the RNA silencing molecule is a small nucleolar RNA (snoRNA). The term "snoRNA" refers to a class of small RNA molecules that primarily direct the chemical modification of other RNAs, such as rRNA, tRNA, and snRNA. snoRNAs typically fall into one of two classes: C / D box snoRNAs, which are typically about 70-120 nucleotides in length and are associated with methylation, and H / ACA box snoRNAs, which are typically about 100-200 nucleotides in length and are associated with pseudouridylation.

[0180] ScaRNAs (i.e., small Cajal body RNA genes), which play a similar role in RNA maturation as snoRNAs, are similar to snoRNAs but target spliceosomal snRNAs, performing site-specific modifications of spliceosomal snRNA precursors (in nuclear Cajal bodies).

[0181] According to one embodiment, the RNA silencing molecule is extracellular RNA (exRNA). The term "exRNA" refers to an RNA species that exists outside the cell in which it was transcribed (e.g., exosomal RNA).

[0182] According to one embodiment, the RNA silencing 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.

[0183] According to certain embodiments, non-limiting examples of RNA molecules that associate with RISC include, but are not limited to, microRNAs (miRNAs), Piwi-binding RNAs (piRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), phased small interfering RNAs (phasiRNAs), trans-acting siRNAs (tasiRNAs), small nuclear RNAs (snRNAs or URNAs), transposable element RNAs (e.g., autonomous and non-autonomous transposable RNAs), transfer RNAs (tRNAs), small nucleolar RNA molecules (snoRNAs), small Cajal body RNAs (scaRNAs), ribosomal RNAs (rRNAs), extracellular RNAs (exRNAs), repeat-derived RNAs, and long non-coding RNAs (lncRNAs).

[0184] According to certain embodiments, non-limiting examples of RNAi molecules that associate with RISC include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-binding RNA (piRNA), phased small interfering RNA (phasiRNA), and trans-acting siRNA (tasiRNA).

[0185] According to one embodiment, the method includes identifying nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology, including but not limited to complete identity, to a nucleic acid sequence encoding an RNA molecule that associates with RISC (e.g., an RNAi-like or miRNA-like sequence).

[0186] According to one embodiment, the RNA molecules of step (a) exhibit a predetermined range of sequence homology, which does not include complete identity, to RNA molecules that associate with RISC and / or are processed into molecules that associate with RISC.

[0187] The term "RNAi-like" refers to a sequence in a genome that has sequence homology to an RNA silencing molecule, but is not identical to the sequence of the RNA silencing molecule.

[0188] The term "miRNA-like" refers to a sequence in the genome that has sequence homology to a miRNA, but is not identical to the miRNA sequence.

[0189] Such non-coding RNA-associated molecules (i.e., miRNA-like molecules) may be functional (e.g., processable and / or have silencing activity, as described below) or dysfunctional (e.g., non-processable or aberrantly processed and / or lack silencing activity, as described below). According to one embodiment, sequence homology ranges include 50% to 99.9%, 60% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, and 95% to 99.9% identity to a nucleic acid sequence encoding an RNA molecule that associates with RISC.

[0190] According to certain embodiments, the sequence homology range comprises 50% to 75% identity to the nucleic acid sequence encoding the RNA molecule that associates with RISC.

[0191] According to certain embodiments, the sequence homology range comprises 50% to 99.9% identity to the nucleic acid sequence encoding the RNA molecule that associates with RISC.

[0192] According to certain embodiments, the sequence homology ranges are sequences encoding RNA molecules that associate with RISC. It contains 70% to 99.9% identity to the nucleic acid sequence.

[0193] According to certain embodiments, the sequence homology range comprises 75% to 99.6% identity to the nucleic acid sequence encoding the RNA molecule that associates with RISC.

[0194] According to certain embodiments, the sequence homology range comprises 85% to 99.6% identity to the nucleic acid sequence encoding the RNA molecule that associates with RISC.

[0195] According to one embodiment, sequence homology comprises 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.6%, or 99.9% identity to a nucleic acid sequence encoding an RNA molecule that associates with RISC.

[0196] According to one embodiment, the sequence homology ranges include 50% to 99.9%, 60% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, 95% to 99.9% identity to a nucleic acid sequence encoding an RNA molecule that associates with and is processed by RISC.

[0197] According to certain embodiments, the sequence homology range comprises 50% to 75% identity to the nucleic acid sequence encoding and processed by the RNA molecule that associates with RISC.

[0198] According to certain embodiments, the sequence homology range comprises 50% to 99.6% identity to the nucleic acid sequence encoding and processed by the RNA molecule that associates with RISC.

[0199] According to certain embodiments, the sequence homology range comprises 70% to 99.9% identity to the nucleic acid sequence encoding and processed by the RNA molecule that associates with RISC.

[0200] According to certain embodiments, the sequence homology range comprises 75% to 99.6% identity to the nucleic acid sequence encoding and processed by the RNA molecule that associates with RISC.

[0201] According to certain embodiments, the sequence homology range comprises 85% to 99.6% identity to the nucleic acid sequence encoding and processed by the RNA molecule that associates with RISC.

[0202] According to one embodiment, sequence homology comprises 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.6%, or 99.9% identity to a nucleic acid sequence encoding and processed by an RNA molecule that associates with RISC.

[0203] According to one embodiment, the sequence homology ranges include 50% to 99.9%, 60% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, and 95% to 99.9% identity to the nucleic acid sequence of a mature RNA silencing molecule that associates with RISC.

[0204] According to certain embodiments, the sequence homology range comprises 50% to 75% sequence homology to the nucleic acid sequence of the mature RNA silencing molecule that associates with RISC.

[0205] According to certain embodiments, the sequence homology range comprises 50% to 99.6% sequence homology to the nucleic acid sequence of a mature RNA silencing molecule that associates with RISC.

[0206] According to certain embodiments, the sequence homology range comprises 70% to 99.9% sequence homology to the nucleic acid sequence of the mature RNA silencing molecule that associates with RISC.

[0207] According to certain embodiments, the sequence homology range comprises 75% to 99.6% sequence homology to the nucleic acid sequence of the mature RNA silencing molecule that associates with RISC.

[0208] According to certain embodiments, the sequence homology range comprises 85% to 99.6% sequence homology to the nucleic acid sequence of the mature RNA silencing molecule that associates with RISC.

[0209] According to one embodiment, the sequence homology comprises 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.6%, or 99.9% identity to the nucleic acid sequence of the mature RNA silencing molecule that associates with RISC.

[0210] According to some embodiments, the phrase "predetermined sequence homology range" as used herein refers to a combination of sequence coverage and sequence homology. As known to those skilled in the art, the term "sequence coverage" refers to the length of a query sequence that contains at least some nucleotides that perfectly match a second sequence, such as a genomic region (e.g., if only the last 90 bases of a 100-base query sequence contain nucleotides that match the second sequence, the coverage is 90%). As known to those skilled in the art, there may be various degrees of homology within the covered sequence (e.g., a sequence with 90% coverage may have a different number of identical nucleotides, different gaps, etc., and therefore different degrees of homology). Sequence coverage and sequence homology can be assessed using any method known in the art; for example, a sequence alignment program such as Blast provides the length of the sequence and the length of the alignment region, from which sequence coverage can be determined.

[0211] According to some embodiments, the predetermined sequence homology range comprises about 50%-100% sequence coverage of the aligned sequences, and in some cases about 70%-100% sequence coverage of the aligned sequences. According to other embodiments, the predetermined sequence homology range comprises about 5%-100%, 25%-100%, 40%-100%, 50%-100%, 70%-100%, or 75%-100% sequence coverage. Each possibility represents a separate embodiment of the present invention.

[0212] According to some embodiments, the predetermined sequence homology range includes: (1) about 50% to 100% of the aligned sequences, optionally about 70% to 100% sequence coverage of the aligned sequences; and (2) about 75% to 100%, optionally about 85% to 100% sequence homology. Each possibility represents a separate embodiment of the invention. According to some embodiments, the predetermined sequence homology range includes at least about 50% coverage with at least about 75% homology.

[0213] According to some embodiments, a nucleic acid sequence encoding an RNA molecule has a predetermined range of sequence homology to a nucleic acid sequence encoding a corresponding silencing RNA (e.g., miRNA) when: (a) the sequence is sequence homologous to the corresponding ncRNA (e.g., miRNA) using default parameters (e.g., www(dot)arabidopsis(dot)org / Blast / BLASToptions(dot)jsp) and (b) the sequence covers at least 50% of the mature sequence of the corresponding silencing RNA (e.g., mature miRNA sequence), which mature sequence is likely to be 19-24 nt in length, likely to be 19-21 nt in length. Each possibility represents a separate embodiment of the present invention.

[0214] According to one embodiment, the sequence homology does not have 100% identity.

[0215] Homology (eg, percent homology, sequence identity plus sequence similarity) can be determined using any homology comparison software that calculates pairwise sequence alignments.

[0216] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to residues in the two sequences that are the same when aligned. When percent sequence identity is used with respect to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thereby not altering the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches to increase the percent sequence identity. Thus, for example, where identical amino acids are assigned a score of 1 and non-conservative substitutions are assigned a score of 0, conservative substitutions are assigned a score between 0 and 1. Scoring of conservative substitutions is 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].

[0217] Identity (e.g., percent homology) can be determined using any homology comparison software, including BlastN software from the National Center of Biotechnology Information (NCBI), such as by using default parameters.

[0218] According to some embodiments of the invention, the identity is a global identity, ie, identity over the entire amino acid or nucleic acid sequence of the invention, and not over a portion thereof.

[0219] According to some embodiments of the invention, the term "homology" or "homologous" refers to the identity of two or more nucleic acid sequences; or the identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequences.

[0220] According to some embodiments of the invention, the homology is global, ie, homology over the entire amino acid or nucleic acid sequence of the invention, and not over a portion thereof.

[0221] The degree of homology or identity between two or more sequences can be determined using a variety of 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.

[0222] When starting with a polynucleotide sequence and comparing it to another polynucleotide sequence, EMB The OSS-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; datafile=EDNAFULL; brief=YES.

[0223] According to some embodiments of the present invention, the parameters used in the EMBOSS-6.0.1 Needleman-Wunsch algorithm are gapopen=10; gapextend=0.2; datafile=EDNAFULL; brief=YES.

[0224] According to some embodiments of the invention, the threshold used to determine homology using the EMBOSS-6.0.1 Needleman-Wunsch algorithm for comparing polynucleotides is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0225] According to some embodiments, determining the degree of homology further requires the use of the Smith-Waterman algorithm (for protein-protein or nucleotide-nucleotide comparisons).

[0226] The default parameters for the GenCore6.0 Smith-Waterman algorithm include: model=sw.model.

[0227] According to some embodiments of the invention, the threshold used to determine 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%.

[0228] According to some embodiments of the present invention, before performing global homology to a polypeptide or polynucleotide of interest (e.g., 80% global homology over the entire sequence), global homology is performed on sequences pre-selected by local homology to the polypeptide or polynucleotide of interest (e.g., 60% identity over 60% of the sequence length). For example, in a first step, homologous sequences are selected using the BLAST software, which uses the Blastp and tBlastn algorithms as filters, and in a second step, alignment is performed using the needle (EMBOSS package) or Frame+ algorithm. Since local identity (Blast alignment) is used only as a filter for the global alignment step, a very permissive cutoff is defined—60% identity over 60% of the sequence length. In this particular embodiment (when using local identity), the default filtering of the Blast package is not utilized (by setting the parameter "-FF").

[0229] In the second step, homologs are defined based on at least 80% global identity to the polypeptide sequence of the core gene. According to some embodiments, the homology is local homology or local identity.

[0230] Local alignment tools include, but are not limited to, the National Center of Biotechnology Information's (NCBI) BlastP, BlastN, BlastX, or TBLASTN software, FASTA, and Smith-Waterman algorithms.

[0231] According to certain embodiments, homology is determined using BlastN version 2.7.1+ with the following default parameters: task=blastn, evalue=10, strand=both, gap opening penalty=5, gap extension penalty=2, match=1, mismatch=-1, word size=11, max scores-25, max alignments=15, query filter=dust, query genetic code-n / a, matrix=no default.

[0232] According to one embodiment, the method further comprises determining the genomic location of nucleic acid sequences encoding RNA molecules that exhibit the predetermined sequence homology range of step (a).

[0233] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (e.g., miRNA-like 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, non-coding RNA genes, enhancers and locus control regions, insulators, S / MAR sequences, non-coding pseudogenes, non-autonomous transposons and retrotransposons, and non-coding simple repeats in the centromeric and telomeric regions of chromosomes.

[0234] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within an intron of a non-coding gene.

[0235] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located in a ubiquitously expressed non-coding gene.

[0236] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located in a non-coding gene that is expressed in a tissue-specific manner.

[0237] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located in an inducibly expressed non-coding gene.

[0238] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located in a developmentally regulated non-coding gene.

[0239] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located between genes, ie, in an intergenic region.

[0240] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located in a coding gene (eg, a protein-coding gene).

[0241] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within an exon of a coding gene (eg, a protein-coding gene).

[0242] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (e.g., miRNA-like molecule) is located within an exon encoding an untranslated region (UTR) of a coding gene (e.g., a protein-coding gene).

[0243] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (e.g., miRNA-like molecule) is located within a translated exon of a coding gene (e.g., a protein-coding gene). Located in.

[0244] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within an intron of a coding gene (eg, a protein-coding gene).

[0245] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within a ubiquitously expressed coding gene.

[0246] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within a coding gene that is expressed in a tissue-specific manner.

[0247] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within an inducibly expressed coding gene.

[0248] According to one embodiment, the nucleic acid sequence encoding the RNAi-like molecule (eg, miRNA-like molecule) is located within a developmentally regulated coding gene.

[0249] According to one embodiment, the method comprises determining the transcription of nucleic acid sequences encoding RNA molecules to select transcribable nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology.

[0250] The phrase "transcribeable nucleic acid sequence" refers to a DNA segment that can be transcribed into RNA.

[0251] Assessment of transcription of nucleic acid sequences can be performed using any method known in the art, for example, by RT-PCR, Northern blot, RNA-seq, small RNA-seq.

[0252] As mentioned above, the methods of some embodiments of the present invention allow for the identification of RNA silencing molecules that can be transcribed but not processed into small RNAs that associate with RISC.

[0253] According to one embodiment, the method comprises determining the processivity into small RNAs of transcripts of transcribable nucleic acid sequences encoding RNA molecules exhibiting a predetermined range of sequence homology to select aberrantly processed (e.g., unprocessable) transcribable nucleic acid sequences encoding RNA molecules exhibiting a predetermined range of sequence homology.

[0254] The term "processing" or "processivity" refers to the biogenesis in which RNA molecules are cleaved into small RNA forms that can associate with RNA-induced silencing complexes (RISCs).Exemplary processing mechanisms include, for example, Dicer and Argonaute, as will be further discussed below.For example, pre-miRNA is processed into mature miRNA, for example, by Dicer.

[0255] The term "canonical processing," as used herein with respect to RNA precursors of a particular class of silencing RNA (e.g., miRNA), refers to the processing of RNA molecules into small RNA molecules in which the processing pattern (e.g., the number, size, and / or location of the resulting small RNA molecules) is typical of precursors of that class of silencing RNA molecules. Typically, the small RNA molecules resulting from canonical processing are capable of associating with RISC and binding to the native target RNA (i.e., the first target RNA). According to some embodiments, wild-type processing as used herein refers to a process that involves ... Reference to a wild-type silencing molecule refers to standard processing. According to some embodiments, reference to a wild-type silencing molecule refers to a standard silencing molecule (i.e., one that behaves, has a structure, and / or is processed according to the known behavior of silencing molecules of that class in the art).

[0256] The term "abnormally processed" as used herein is a comparative term and refers to the processing of RNA molecules into small RNA molecules, such that the processing is not standard processing for the RNA precursor of a particular class of silencing RNA (for example, miRNA).In a non-limiting example, an RNA molecule that is homologous to the precursor of a particular class of silencing RNA molecule (for example, the precursor of miRNA) is processed differently from its precursor (standard processing), and is abnormally processed.

[0257] According to some embodiments, aberrantly processed is selected from the group consisting of not processed (i.e., no small RNA molecules are produced) and differentially processed compared to standard processing (i.e., processed into small RNA molecules of a different number, size, and / or location than achieved by standard processing). Small RNA molecules resulting from aberrant processing are typically of an abnormal size (compared to small RNA molecules obtained from standard processing), do not associate with RISC, and / or are not complementary to their native target RNA (i.e., the first target RNA). Each possibility represents a separate embodiment of the present invention.

[0258] As used herein, the term "small RNA form" or "small RNA" or "small RNA molecule" refers to a mature small RNA that is capable of hybridizing to a target RNA (or a fragment thereof).

[0259] As used herein, the phrase "dysfunctional RNA molecule" refers to an RNA molecule (e.g., a non-coding RNA molecule, e.g., an RNAi molecule) that is not processed into a small RNA that can associate with RISC and does not silence a natural target RNA (i.e., a first target RNA). According to one embodiment, the dysfunctional RNA molecule comprises a sequence change (e.g., a sequence change in a precursor sequence) that alters its secondary RNA structure and causes it to be abnormally processed (e.g., unable to be processed).

[0260] According to one embodiment, the small RNA form has silencing activity.

[0261] According to one embodiment, the small RNA has a length of 250 nucleotides or less, and contains, for example, 15 to 250, 15 to 200, 15 to 150, 15 to 100, 15 to 50, 15 to 40, 15 to 30, 15 to 25, 15 to 20, 20 to 30, 20 to 25, 30 to 100, 30 to 80, 30 to 60, 30 to 50, 30 to 40, 30 to 35, 50 to 150, 50 to 100, 50 to 80, 50 to 70, 50 to 60, 100 to 250, 100 to 200, 100 to 150, 150 to 250, or 150 to 200 nucleotides.

[0262] According to certain embodiments, the small RNA molecule comprises between 20 and 50 nucleotides.

[0263] According to a particular embodiment, the small RNA molecule comprises 20 to 30 nucleotides.

[0264] According to certain embodiments, the small RNA molecule comprises between 21 and 29 nucleotides.

[0265] According to a particular embodiment, the small RNA molecule comprises 21 to 23 nucleotides.

[0266] According to a particular embodiment, the small RNA molecule comprises 21 nucleotides.

[0267] According to a particular embodiment, the small RNA molecule comprises 22 nucleotides.

[0268] According to a particular embodiment, the small RNA molecule comprises 23 nucleotides.

[0269] According to a particular embodiment, the small RNA molecule comprises 24 nucleotides.

[0270] According to a particular embodiment, the small RNA molecule comprises 25 nucleotides.

[0271] According to a particular embodiment, the small RNA molecule consists of 20 to 50 nucleotides.

[0272] According to a particular embodiment, the small RNA molecule consists of 20 to 30 nucleotides.

[0273] According to a particular embodiment, the small RNA molecule consists of 21 to 29 nucleotides.

[0274] According to a particular embodiment, the small RNA molecule consists of 21 to 23 nucleotides.

[0275] According to a particular embodiment, the small RNA molecule consists of 21 nucleotides.

[0276] According to a particular embodiment, the small RNA molecule consists of 22 nucleotides.

[0277] According to a particular embodiment, the small RNA molecule consists of 23 nucleotides.

[0278] According to a particular embodiment, the small RNA molecule consists of 24 nucleotides.

[0279] According to a particular embodiment, the small RNA molecule consists of 25 nucleotides.

[0280] Typically, processivity depends on the structure of the RNA molecule, also referred to herein as structural originality, i.e., the secondary RNA structure (i.e., base-pairing profile). Secondary RNA structure is important for accurate and efficient processing of RNA molecules into small RNAs (such as siRNAs or miRNAs), which is structure-dependent rather than purely sequence-dependent.

[0281] Thus, according to one embodiment, the selected or identified nucleic acid sequence encoding the RNA molecule of step (a) is homologous to a gene encoding a silencing RNA molecule whose silencing activity and / or processing into small silencing RNAs depends on its secondary structure.

[0282] According to some embodiments, silencing RNA molecules whose silencing activity and / or processing into small silencing RNAs depend on secondary structure include microRNAs (miRNAs), short hairpin RNAs (shRNAs), small nuclear RNAs (snRNAs or U-RNAs), small nucleolar RNAs (snoRNAs), small Cajal body RNAs (scaRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), RNAs derived from repeats, RNAs derived from autonomous and non-autonomous transposable and retrotransposable elements, RNAs of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNAs (ln cRNA).

[0283] According to one embodiment, the intracellular RNAi processing machinery, i.e., intracellular RNAi processing and execution factors, processes the RNA molecules into small RNAs.

[0284] According to one embodiment, the intracellular RNAi processing machinery comprises 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 cleavage enzymes (e.g., RNY1, ANGIOGENIN, Rnase P, Rnase P-like, SLFN3, ELAC1, and ELAC2), and Piwi-binding RNA (piRNA)-associated proteins (e.g., AGO3, AUBERGINE, HIWI, HIWI2, HIWI3, PIWI, ALG1, and ALG2).

[0285] According to one embodiment, the intracellular RNAi processing machinery generates an RNA silencing molecule, but the specific target has not been identified.

[0286] According to one embodiment, the small RNA molecules are processed from precursors.

[0287] According to one embodiment, small RNA molecules are processed from single-stranded RNA (ssRNA) precursors.

[0288] According to one embodiment, small RNA molecules are processed from double-stranded single-stranded RNA precursors.

[0289] According to one embodiment, small RNA molecules are processed from unstructured RNA precursors.

[0290] According to one embodiment, small RNA molecules are processed from RNA precursors that encode proteins.

[0291] According to one embodiment, the small RNA molecules are processed from non-coding RNA precursors.

[0292] According to one embodiment, small RNA molecules are processed from dsRNA precursors (eg, containing perfect and imperfect base pairing).

[0293] According to one embodiment, the dsRNA may be derived from two different, complementary RNAs, or may be derived from a single RNA that folds back on itself to form the dsRNA.

[0294] Assessment of processing can be performed using any method known in the art, for example, by smallRNAseq, Northern blot, smallRNAqRT-PCR, rapid amplification of cDNA ends (RACE).

[0295] For example, smallRNAseq, Northern blot, smallRNAqRT-PCR, and rapid amplification of cDNA ends (RACE) methods can be applied to select aberrantly processed (e.g., unprocessable) nucleic acid sequences.

[0296] Functional processivity can also be determined by comparative structural analysis. For example, a dysfunctional pre-miRNA-like structure is compared with a corresponding pre-miRNA that can be processed into a small RNA molecule that associates with RISC (e.g., precursor structures are compared). Alterations in the dysfunctional structure suggest that the pre-miRNA is not processed or is processed differently from the corresponding pre-miRNA that can be processed into a small RNA molecule that associates with RISC. Processing can be verified by small RNA analysis.

[0297] According to one embodiment, steps (b) and / or (c) are affected by aligning the small RNA expression data to the genome of the cell and determining the amount of reads that map to each genomic location.

[0298] According to some embodiments, analyzing small RNAs to determine processing involves aligning the sequences of small RNAs expressed in a particular cell or tissue to their corresponding genomic locations (e.g., within genes encoding potential dysfunctional pre-miRNA-like molecules) to determine the location at which each sRNA is expressed and the number of sRNA reads at each location. According to certain embodiments, the alignment of the sequences of expressed small RNAs to their corresponding genomic locations (i.e., predetermined locations) to determine processing is a mismatch-free alignment.

[0299] As described above, aberrantly processed transcribable nucleic acid sequences are selected that encode RNA molecules that exhibit a predetermined range of sequence homology.

[0300] According to one embodiment, the method includes modifying the nucleic acid sequence of an aberrantly processed (e.g., unprocessable) transcribable nucleic acid sequence to associate with RISC and confer processibility to a small RNA complementary to a first target RNA (e.g., a naturally occurring target RNA, as described below), also referred to herein as "reactivating" silencing activity.

[0301] According to one embodiment, the modification in step (d) comprises introducing into the cell a DNA editing agent that reactivates the silencing activity of the aberrantly processed RNA molecule toward the first target RNA, thereby generating an RNA molecule with silencing activity in the cell.

[0302] According to one embodiment, the method further includes altering the specificity of the RNA molecule having silencing activity in the cell, wherein the DNA editing agent redirects the silencing specificity of the RNA molecule toward a target RNA of interest that is different from the first target RNA, thereby altering the specificity of the RNA molecule having silencing activity in the cell.

[0303] According to one embodiment, the difference between modifying to activate silencing for the first target RNA and modifying specificity can be the use of different GEiGS oligos when performing GEiGS (i.e., GEiGS oligos for modifying specificity further include modifying the sequence of the mature miRNA to change the specificity).

[0304] Below are descriptions of various non-limiting examples of methods and DNA editing agents used to introduce nucleic acid changes into genes encoding RNA silencing molecules, as well as agents for doing so that can be used in accordance with certain embodiments of the present disclosure.

[0305] Genome editing using engineered endonucleases - This approach uses artificially engineered nucleases to typically cut at desired location(s) in the genome, creating specific double-strand breaks (DSBs), which are then transduced by homologous recombination (HR) or This refers to a reverse genetic method that repairs DNA using endogenous processes in cells, such as non-homologous end joining (NHEJ). NHEJ directly joins the DNA ends of a double-strand break (DSB) with or without minimal end trimming, while HR utilizes a homologous donor sequence as a template (i.e., a sister chromatid formed during S phase) to regenerate / copy the missing DNA sequence at the break site. To introduce specific nucleotide modifications into genomic DNA, a donor DNA repair template (exogenously provided single-stranded or double-stranded DNA) containing the desired sequence must be present during HR.

[0306] Genome editing cannot be performed using conventional restriction endonucleases because most restriction enzymes target a few base pairs in DNA, and these sequences are often found in many locations throughout the genome, resulting in multiple cuts that are not limited to the desired location. To overcome this challenge and create site-specific single- or double-strand breaks (DSBs), several different classes of nucleases have been discovered and bioengineered. These include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas9 system.

[0307] Meganucleases - Meganucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys Box family, and the HNH family. These families are characterized by structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases differ significantly from each other with respect to conserved structural elements and, therefore, DNA recognition sequence specificity and catalytic activity. Meganucleases are commonly found in microbial species and have the unique property of having very long recognition sequences (>14 bp), which allows for very high specificity for cleavage at the natural, desired site.

[0308] This can be used to create site-specific double-strand breaks (DSBs) in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. To overcome this problem, using mutagenesis and high-throughput screening methods, meganuclease variants that recognize unique sequences have been created. For example, various meganucleases have been fused to create hybrid enzymes that recognize new sequences.

[0309] Alternatively, the DNA-interacting amino acids of the meganuclease can be altered to design sequence-specific meganucleases (see, e.g., U.S. Patent No. 8,021,867). Meganucleases can be designed using methods described, for example, 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, the contents of each of which are incorporated herein by reference in their entirety. Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technologies, such as Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0310] ZFNs and TALENs - Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are two different classes of engineered nucleases. All of these nucleases have proven effective in creating 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).

[0311] Essentially, ZFN and TALEN restriction endonuclease technologies utilize a nonspecific DNA-cleaving enzyme linked to a specific DNA-binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme is selected whose DNA recognition and cleavage sites are separated from each other. The cleavage portion is then ligated to the DNA-binding domain, thereby resulting in an endonuclease with extremely high specificity for the desired sequence. An exemplary restriction enzyme with such properties is Fokl. Furthermore, Fokl has the advantage of requiring dimerization for nuclease activity, meaning that specificity is dramatically enhanced when each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered to function only as heterodimers and have high catalytic activity. Nucleases that function as heterodimers avoid the potential for unwanted homodimer activity, thereby enhancing the specificity of double-strand breaks (DSBs).

[0312] Thus, for example, to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind to adjacent sequences at the target site. When transiently expressed in cells, the nuclease binds to its target site, and the FokI domain heterodimerizes to create double-strand breaks (DSBs). Repair of these double-strand breaks (DSBs) through the non-homologous end joining (NHEJ) pathway often results in small deletions or small sequence insertions (indels). Because each repair performed by NHEJ is unique, a single nuclease pair can be used to create a variety of alleles with different insertions or deletions at the target site.

[0313] Generally, NHEJ is relatively accurate (approximately 75-85% of DSBs in human cells are repaired by NHEJ within approximately 30 minutes of detection). In gene editing, if repair were accurate, we would rely on incorrect NHEJ because the nuclease would continue to cleave until the repair product becomes mutagenic and the recognition / cleavage site / PAM motif is lost / mutated, or until the transiently introduced nuclease is no longer present.

[0314] Typically, deletions range in length from a few base pairs to several hundred 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, introducing a DNA fragment homologous to the target region in conjunction with the nuclease pair can repair double-strand breaks (DSBs) via homologous recombination (HR) (e.g., in the presence of a donor template) to generate specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0315] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, and TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic of naturally occurring in combination in proteins. Cys2-His2 zinc fingers are typically found in repeats spaced 3 bp apart, and are found in various combinations in various nucleic acid-interacting proteins. On the other hand, TALEs are found in repeats with a 1:1 recognition ratio between the amino acid and the recognized nucleotide pair. Both zinc fingers and TALEs occur in a repetitive pattern. Therefore, various combinations can be tried to generate diverse sequence specificities. Approaches to generating site-specific zinc finger endonucleases include, for example, modular assembly (arrangement and binding of zinc fingers correlated with triplet sequences to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides, followed by high stringency selection of peptide combinations versus final targets in a bacterial system), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs are designed, for example, at Sangamo Biosciences™ (Richmond, CA), and are also commercially available.

[0316] Methods for designing and obtaining TALENs are described, for example, in Reyon et al. Nature Biotechnology 2012 May;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 named Mojo Hand has been introduced by Mayo Clinic to design TAL and TALEN constructs for genome editing applications (accessible through www(dot)talendesign(dot)org). TALENs are designed, for example, by Sangamo Biosciences™ (Richmond, CA), and can also be commercially obtained.

[0317] T-GEE System (Target Gene Genome Editing Engine) - A programmable nucleoprotein molecular complex containing a polypeptide moiety and a specificity-conferring nucleic acid (SCNA) is provided that can assemble in vivo in a target cell and interact 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 including (i) a functional domain capable of modifying the target site and (ii) a linking domain capable of interacting with the specificity-conferring nucleic acid, and (b) a specificity-conferring nucleic acid (SCNA) that includes (i) a nucleotide sequence complementary to a region of the target nucleic acid adjacent to the target site and (ii) a recognition region capable of specifically binding to the linking domain of the polypeptide. Through base pairing between the specificity-conferring nucleic acid and the target nucleic acid, the composition enables precise, reliable, and cost-effective modification of a predetermined nucleic acid sequence target with high specificity and the binding ability of the molecular complex to the target nucleic acid. The compositions have low genotoxicity, are modularly assembled, utilize a single platform without customization, are practical for independent use outside of specialized core facilities, have short development times, and are low cost.

[0318] CRISPR-Cas systems and all their variants (also referred to herein as "CRISPR")—Many bacteria and archaea contain endogenous RNA-based adaptive immune systems capable of degrading the nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeats (CRISPR) nucleotide sequences that generate the RNA components and CRISPR-associated (Cas) genes that encode the protein components. CRISPR RNA (crRNA) contains short stretches of homology to specific viral and plasmid DNA and acts as a guide to instruct Cas nucleases to degrade complementary nucleic acids in the corresponding pathogens. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that three components—Cas9 nuclease, crRNA containing 20 base pairs of homology to the target sequence, and trans-activating crRNA (tracrRNA)—are involved in the RNA / Cas system. They form a protein complex that, together, has been shown to be sufficient for sequence-specific nuclease activity (Jinek et al. Science (2012) 337:816-821).

[0319] Furthermore, it has been demonstrated that synthetic chimeric guide RNAs (sgRNAs) composed of a fusion between crRNA and tracrRNA can direct Cas9 to cleave DNA targets complementary to the crRNA in vitro. It has also been demonstrated that transient expression of Cas9 in conjunction with synthetic sgRNAs can be used to generate targeted 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). al., 2013).

[0320] The CRISPR / Cas system for genome editing contains two distinct components: an sgRNA and an endonuclease, such as Cas9.

[0321] The sgRNA (also referred to herein as short guide RNA (sgRNA)) is typically a 20-nucleotide sequence encoding a target homologous sequence (crRNA) in a single chimeric transcript, combined with an endogenous bacterial RNA (tracrRNA) that links the crRNA to the Cas9 nuclease. The gRNA / Cas9 complex is recruited to the target sequence through base pairing between the sgRNA sequence and complementary genomic DNA. For Cas9 to successfully bind, the target sequence must also contain the correct protospacer adjacent motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas9 complex localizes Cas9 to the target sequence so that it can cleave both strands of DNA, creating a double-strand break (DSB). Similar to ZFNs and TALENs, the double-strand break (DSB) generated by CRISPR / Cas can undergo homologous recombination or NHEJ and is susceptible to specific sequence modification during DNA repair.

[0322] The Cas9 nuclease has two functional domains, RuvC and HNH, which each cleave a different DNA strand. When both of these domains are active, Cas9 creates a double-strand break (DSB) in genomic DNA.

[0323] A major advantage of CRISPR / Cas is that the system combines high efficiency with the ability to easily generate synthetic sgRNAs. This provides a system that can be easily modified to target modifications at different genomic sites and / or to target different modifications at the same site. Furthermore, protocols have been established that allow for simultaneous targeting of multiple genes. The majority of cells carrying mutations have biallelic mutations in the targeted genes.

[0324] However, the apparent flexibility in base-pairing interactions between the sgRNA sequence and the target sequence in genomic DNA allows Cas9 to cleave even imperfect matches to the target sequence.

[0325] Engineered versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called "nickases." Because they have only one active nuclease domain, Cas9 nickases cleave only one strand of the target DNA, creating a single-strand break or "nick." The single-strand break or nick is repaired mostly by single-strand break repair mechanisms involving proteins such as, but not limited to, PARP (sensor) and the XRCC1 / LIG III complex (ligation). Single-strand breaks can be repaired by topoisomerase I poisons or drugs that capture PARP1 in naturally occurring SSBs. If double-strand breaks (SSBs) are generated, they may persist and become single-ended DSBs that can only be repaired by HR when cells enter S phase and a replication fork encounters such an SSB. However, two adjacent nicks on opposite strands introduced by Cas9 nickase are treated as double-strand breaks and are often referred to as "double-nick" CRISPR systems. Double nicks are essentially nonparallel DSBs and, like other DSBs, can be repaired by HR or NHEJ, depending on the desired effect on the gene target and the presence of donor sequences and the stage of the cell cycle (HR is much less abundant and can only occur in the S and G2 phases of the cell cycle). Therefore, when reduction of off-target effects and specificity are important, using Cas9 nickase to create double nicks by designing two sgRNAs with target sequences on opposite strands of genomic DNA in close proximity would reduce, if not eliminate, off-target effects, because it would create a nick that would not be able to alter genomic DNA by either sgRNA alone.

[0326] A modified version of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9 or dCas9) lacks nuclease activity but can still bind to DNA based on the specificity of the sgRNA. dCas9 can be used as a platform for DNA transcription regulators to activate or repress gene expression by fusing the inactive enzyme to a known regulatory domain. For example, binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

[0327] Additional variants of Cas9 that can be used in some embodiments of the present invention include, but are not limited to, CasX and Cpf1. The CasX enzyme is smaller in size than Cas9 and comprises a distinct family of RNA-guided genome editors found in bacteria (typically not in humans), making it less likely to provoke an immune system response in humans. CasX also utilizes a different PAM motif than Cas9 and can be used to target sequences lacking the Cas9 PAM motif (see Liu JJ et al., Nature. (2019) 566(7743):218-223). Cpf1, also referred to as Cas12a, is particularly advantageous for editing AT-rich regions, where the Cas9 PAM (NGG) is much less abundant (see Li T et al., Biotechnol Adv. (2019) 37(1):21-27; Murugan K et al., Mol Cell. (2017) 68(1):15-25).

[0328] According to another embodiment, the CRISPR system may be fused to various effector domains, such as a DNA cleavage domain. The DNA cleavage domain can be derived from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which the DNA cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases (e.g., New England (See Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.). In exemplary embodiments, the cleavage domain of the CRISPR system is a Fokl endonuclease domain or a modified Fokl endonuclease domain. Another option is the use of homing endonucleases (HEs). HEs are small proteins (<300 amino acids) found in bacteria, archaea, and unicellular eukaryotes. A distinctive feature of HEs is that they recognize relatively long sequences (14-40 bp) compared to other site-specific endonucleases such as restriction enzymes (4-8 bp). Hes have historically been classified by 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 related to EdxHD enzymes and are considered by some to be separate families. At the structural level, the HNH and His-Cys Boxes are similar to those found in PD-(D / E)xK and EdxHD enzymes. They share a common fold (designated ββα-metal). Each family has different catalytic and DNA recognition strategies, making them suitable to varying degrees for genetic engineering for various applications. See, for example, Methods Mol Biol. (2014) 1123:1-26. Exemplary homing endonucleases that can be used in accordance with some embodiments of the present invention include, but are not limited to, I-CreI, I-TevI, I-HmuI, I-PpoI, and I-Ssp68031.

[0329] Modified versions of CRISPR, such as dead CRISPR (dCRISPR endonuclease), can also be used to inhibit CRISPR transcription (CRISPRi) or activate CRISPR transcription (CRISPRa). See, e.g., Kampmann M., ACS Chem Biol. (2018) 13(2):406-416; La Russa MF and Qi LS., Mol Cell Biol. (2015) 35(22):3800-9.

[0330] Other versions of CRISPR that can be used in accordance with some embodiments of the invention include genome editing, which uses components of the CRISPR system, along with other enzymes, to create point mutations directly in a cell's DNA or RNA.

[0331] Thus, according to one embodiment, the editing agent is a DNA or RNA editing agent.

[0332] According to one embodiment, the DNA or RNA editing agent induces base editing.

[0333] The term "base editing," as used herein, refers to introducing point mutations into a cell's DNA or RNA without creating double- or single-stranded DNA breaks.

[0334] In base editing, DNA base editors typically contain a catalytically impaired Cas nuclease fused to a base-modifying enzyme that acts on single-stranded DNA (ssDNA). Upon binding to its target DNA locus, the gRNA base-pairs with the target DNA strand, displacing a small segment of single-stranded DNA in an "R-loop." The DNA bases within this ssDNA bubble are then modified by a base-editing enzyme (e.g., a deaminase enzyme). To improve efficiency in eukaryotic cells, the catalytically impaired nuclease also creates nicks in the unedited DNA strand, inducing the cell to repair the unedited strand using the edited strand as a template.

[0335] Two classes of DNA base editors have been reported: cytosine base editors (CBEs), which convert CG base pairs to TA base pairs, and adenine base editors (ABEs), which convert AT base pairs to GC base pairs. Collectively, CBEs and ABEs can mediate all four possible pairwise mutations (C → T, A → G, T → C, and G → A). Similarly, in RNA, targeted adenosine-to-inosine conversion utilizes both antisense and Cas13-guided RNA targeting methods.

[0336] According to one embodiment, the DNA or RNA editing agent comprises a catalytically inactive endonuclease (e.g., CRISPR-dCas).

[0337] According to one embodiment, the catalytically inactive endonuclease is an inactive Cas9 (e.g., dCas9).

[0338] According to one embodiment, the catalytically inactive endonuclease is an inactive Cas13 (e.g., dCas13).

[0339] According to one embodiment, the DNA or RNA editing agent comprises an enzyme capable of epigenetic editing (i.e., making chemical changes to DNA, RNA, or histone proteins).

[0340] Exemplary enzymes include, but are not limited to, DNA methyltransferases, methylases, and acetyltransferases. More specifically, exemplary enzymes include, for example, DNA (cytosine-5) methyltransferase 3A (DNMT3a), histone acetyltransferase p300, 10-11 translocation methylcytosine dioxygenase 1 (TET1), lysine (K)-specific demethylase 1A (LSD1), and calcium and integrin binding protein 1 (CIB1).

[0341] In addition to catalytically deficient nucleases, the DNA or RNA editing agents of the present invention may also include nucleobase deaminase enzymes and / or DNA glycosylase inhibitors.

[0342] According to certain embodiments, the DNA or RNA editing agent comprises BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), or BE3 (APOBEC-XTEN-dCas9(A840H)-UGI) in conjunction with an sgRNA. APOBEC1 is the full-length or catalytically active fragment of the deaminase, XTEN is a protein linker, UGI is a uracil DNA glycosylase inhibitor that prevents subsequent repair of the U:G mismatch back to a C:G base pair, and dCas9(A840H) is a nickase that nicks only the unedited strand, restoring catalytic activity of the HNH domain and restoring dCas9 to its original state to prime newly synthesized DNA and produce the desired U:A product.

[0343] Additional enzymes that can be used for base editing according to some embodiments of the present invention are set forth in Rees and Liu, Nature Reviews Genetics (2018) 19:770-788, which is incorporated herein by reference in its entirety.

[0344] In addition to the tools available to aid in the selection and / or design of target sequences, numerous bioinformatically determined lists of sgRNAs specific to different genes in different species are publicly available, including, but not limited to, Target Finder from the Feng Zhang Laboratory, Target Finder (E-CRISP) from the Michael Boutros Laboratory, RGEN Tools:Cas-OFFinder, CasFinder: A Flexible Algorithm for Identifying Specific Cas9 Targets in Genomes, and CRISPR Optimal Target Finder.

[0345] To use the CRISPR system, both the sgRNA and the Cas endonuclease (e.g., Cas9, Cpf1, CasX) must be expressed or present (e.g., as a ribonucleoprotein complex) in the target cell. The insertion vector may contain both cassettes on one plasmid, or the cassettes are expressed from two separate plasmids. CRISPR plasmids are available from Addgene (75 Sydney). Plasmids such as the px330 plasmid from Sigma-Aldrich, Inc. (St., Suite 550A, Cambridge, MA 02139) are commercially available. The use of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) guide RNA technology and Cas endonucleases to modify plant genomes is also 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 U.S. Patent Application Publication No. 20150082478, the entire contents of which are specifically incorporated herein by reference. Use of sgRNAs to perform DNA editing Cas endonucleases that can achieve this include, but are not limited to, Cas9, Cpf1, CasX (Zetsche et al., 2015, Cell. 163(3):759-71), C2c1, C2c2, and C2c3 (Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97).

[0346] "Hit-and-run" or "in-out" recombination involves a two-step recombination procedure. In the first step, the desired sequence change is introduced using an insertion vector containing a dual positive / negative selectable marker cassette. The insertion vector contains a single contiguous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at one site within the homologous region and introduced into cells, where positive selection is performed to isolate homologous recombination-mediated events. DNA containing the homologous sequence can be provided as a plasmid or as a single- or double-stranded oligo. These homologous recombinants contain local duplications separated by intervening vector sequences containing the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the overlapping sequences. The duplication is removed by a local recombination event, and depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild-type. Ultimately, the desired modifications are introduced without retaining any exogenous sequences.

[0347] The "double replacement" or "tag and exchange" strategy involves a two-step selection procedure similar to the hit-and-run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a double positive / negative selectable cassette adjacent to the desired mutation site. After introducing the system components into cells and applying positive selection, HR-mediated events can be identified. Next, a second targeting vector containing a region homologous to the desired mutation is introduced into the targeted clone, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation but excludes unwanted exogenous sequences.

[0348] According to certain embodiments, the DNA editing agent comprises a DNA targeting module (e.g., a gRNA).

[0349] According to certain embodiments, the DNA editing agent does not comprise an endonuclease.

[0350] According to certain embodiments, the DNA editing agent comprises an endonuclease.

[0351] According to certain embodiments, the DNA editing agent comprises a catalytically inactive endonuclease.

[0352] According to certain embodiments, the DNA editing agent comprises a nuclease (e.g., an endonuclease) and a DNA targeting module (e.g., an sgRNA).

[0353] According to certain embodiments, the DNA editing agent is a CRISPR / endonuclease.

[0354] According to certain embodiments, the DNA editing agent is CRISPR / Cas, e.g., sgRNA and Cas9, or sgRNA and dCas9.

[0355] According to certain embodiments, the DNA editing agent is CRISPR / Cas9, for example, as disclosed in WO 2019 / 058255, the entire contents of which are incorporated herein by reference.

[0356] In certain embodiments, the DNA or RNA editing agent induces base editing.

[0357] According to certain embodiments, the DNA or RNA editing agent comprises an enzyme for epigenetic editing.

[0358] According to certain embodiments, the DNA editing agent is a TALEN.

[0359] According to certain embodiments, the DNA editing agent is a ZFN.

[0360] According to certain embodiments, the DNA editing agent is a meganuclease.

[0361] According to one embodiment, the DNA editing agent is linked to a reporter to monitor expression within a cell (e.g., a eukaryotic cell).

[0362] According to one embodiment, the reporter is a fluorescent reporter protein.

[0363] The term "fluorescent protein" refers to a polypeptide that emits fluorescence, typically detectable by flow cytometry, microscopy, or any fluorescent imaging system, and can therefore be used as the basis for selection of cells expressing such a protein.

[0364] Examples of fluorescent proteins that can be used as reporters include, but are not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (e.g., dsRed, mCherry, RFP). A non-limiting list of fluorescent or other reporters includes proteins that are detectable by luminescence (e.g., luciferase) or colorimetric assays (e.g., GUS). According to certain embodiments, the fluorescent reporter is a red fluorescent protein (e.g., dsRed, mCherry, RFP) or GFP.

[0365] A review of new classes of fluorescent proteins and applications 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].

[0366] According to another embodiment, the reporter is an endogenous gene of the plant. An exemplary reporter is the phytoene desaturase gene (PDS3), which encodes one of the key enzymes in the carotenoid biosynthesis pathway. Its silencing results in an albino / albino phenotype. Thus, plants with reduced PDS3 expression have reduced chlorophyll levels, leading to complete albino and dwarfism. Additional genes that can be used in accordance with the present teachings include, but are not limited to, genes involved in crop protection.

[0367] According to another embodiment, the reporter is an antibiotic selection marker. Examples of antibiotic selection markers that can be used as reporters are neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt). Additional marker genes that can be used in accordance with the present teachings include, but are not limited to, gentamicin acetyltransferase (accC3) resistance and bleomycin and phleomycin resistance genes.

[0368] It will be appreciated that the enzyme NPTII inactivates many aminoglycoside antibiotics, such as kanamycin, neomycin, genetin (or G418), and paromomycin, by phosphorylation, of which kanamycin, neomycin, and paromomycin are used in a variety of plant species, and G418 is routinely used for the selection of transformed mammalian cells.

[0369] According to another embodiment, the reporter is a toxic selectable marker. An exemplary toxic selectable marker that can be used as a reporter is, but is not limited to, allyl alcohol selection using the alcohol dehydrogenase (ADH1) gene. ADH1, which contains a group of dehydrogenase enzymes that catalyze the interconversion between alcohol and aldehydes or ketones while simultaneously reducing NAD+ or NADP+, degrades alcoholic toxic substances in tissues. Plants with reduced expression of ADH1 exhibit increased tolerance to allyl alcohol. Therefore, plants with reduced ADH1 are resistant to the toxic effects of allyl alcohol.

[0370] Regardless of the DNA editing agent used, the methods of the invention are used such that a gene encoding an aberrantly processed (e.g., non-processable) transcribable RNA silencing molecule is modified by at least one of a deletion, an insertion, or a point mutation.

[0371] According to one embodiment, the structured region of the RNA silencing molecule is modified.

[0372] According to one embodiment, the stem region of the RNA silencing molecule is modified.

[0373] According to one embodiment, the loop region of the RNA silencing molecule is modified.

[0374] According to one embodiment, the stem and loop regions of the RNA silencing molecule are modified.

[0375] According to one embodiment, the unstructured region of the RNA silencing molecule is modified.

[0376] According to one embodiment, the stem and loop regions and the unstructured regions of the RNA silencing molecule are modified.

[0377] According to one embodiment, nucleic acid sequence modifications of a transcribable nucleic acid sequence encoding an aberrantly processed RNA molecule exhibiting a predetermined range of sequence homology affect a nucleic acid other than that corresponding to the binding site with a first target RNA (e.g., a natural target RNA), e.g., a nucleic acid other than that encoding the mature sequence of an RNAi capable of binding to the natural target.

[0378] According to one embodiment, the modification confers processibility of the RNA silencing molecule into small RNAs that associate with RISC.

[0379] According to certain embodiments, the modification is about 1 to 500 nucleotides, about 1 to 250 nucleotides, about 1 to 150 nucleotides, about 1 to 100 nucleotides, about 1 to 50 nucleotides, about 1 to 200 nucleotides, about 1 to 3 ... The amino acid sequence includes modifications of 25 nucleotides, about 1 to 10 nucleotides, 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.

[0380] According to one embodiment, the modification comprises modification of at most 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, 250, 300, 350, 400, 450, or at most 500 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule).

[0381] According to one embodiment, a contiguous nucleic acid sequence (e.g., at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500 bases) may be modified.

[0382] According to one embodiment, for example, 20, 50, 100, 150, 200, 500, 1000, 2000, 5000 nucleic acids may be non-contiguously modified throughout the sequence.

[0383] According to particular embodiments, the modification comprises modification of up to 200 nucleotides.

[0384] According to particular embodiments, the modification comprises modification of up to 150 nucleotides.

[0385] According to particular embodiments, the modification comprises modification of up to 100 nucleotides.

[0386] According to particular embodiments, the modification comprises modification of up to 50 nucleotides.

[0387] According to particular embodiments, the modification comprises modification of up to 25 nucleotides.

[0388] According to particular embodiments, the modification comprises modification of up to 24 nucleotides.

[0389] According to particular embodiments, the modification comprises modification of up to 23 nucleotides.

[0390] According to particular embodiments, the modification comprises modification of up to 22 nucleotides.

[0391] According to particular embodiments, the modifications comprise modifications of up to 21 nucleotides.

[0392] According to particular embodiments, the modification comprises the modification of up to 20 nucleotides.

[0393] According to particular embodiments, the modification comprises modification of up to 15 nucleotides.

[0394] According to particular embodiments, the modification comprises the modification of up to 10 nucleotides.

[0395] According to particular embodiments, the modification comprises modification of up to 5 nucleotides.

[0396] According to one embodiment, the recognition / cleavage site / PAM motif of the RNA silencing molecule is modified to eliminate the original PAM recognition site.

[0397] According to certain embodiments, at least 1, 2, 3, 4, 5, 6 in the PAM motif , 7, 8, 9, 10, or more nucleic acids may be modified.

[0398] According to one embodiment, the modification comprises an insertion.

[0399] According to certain embodiments, the insertion comprises an insertion of about 1 to 500 nucleotides, about 1 to 250 nucleotides, about 1 to 150 nucleotides, about 1 to 100 nucleotides, about 1 to 50 nucleotides, about 1 to 25 nucleotides, about 1 to 10 nucleotides, 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 aberrantly processed transcribable RNA silencing molecule).

[0400] According to one embodiment, the insertion comprises an insertion of at most 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, 250, 300, 350, 400, or at most 500 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule).

[0401] According to particular embodiments, the insertion comprises an insertion of up to 200 nucleotides.

[0402] According to particular embodiments, the insertion comprises an insertion of up to 150 nucleotides.

[0403] According to particular embodiments, the insertion comprises an insertion of at most 100 nucleotides.

[0404] According to certain embodiments, the insertion comprises an insertion of up to 50 nucleotides.

[0405] According to certain embodiments, the insertion comprises an insertion of up to 25 nucleotides.

[0406] According to certain embodiments, the insertion comprises an insertion of up to 24 nucleotides.

[0407] According to certain embodiments, the insertion comprises an insertion of up to 23 nucleotides.

[0408] According to certain embodiments, the insertion comprises an insertion of up to 22 nucleotides.

[0409] According to certain embodiments, the insertion comprises an insertion of up to 21 nucleotides.

[0410] According to certain embodiments, the insertion comprises an insertion of at most 20 nucleotides.

[0411] According to certain embodiments, the insertion comprises an insertion of at most 15 nucleotides.

[0412] According to particular embodiments, the insertion comprises an insertion of at most 10 nucleotides.

[0413] According to particular embodiments, the insertion comprises an insertion of at most 5 nucleotides.

[0414] According to one embodiment, the modification comprises a deletion.

[0415] According to certain embodiments, the deletion is a deletion of a transcriptional sequence (a transcriptional sequence of an abnormally processed transcriptional sequence) or a deletion of a transcriptional sequence (a transcriptional sequence of an abnormally processed transcriptional sequence). The deletions include deletions of about 1 to 500 nucleotides, about 1 to 250 nucleotides, about 1 to 150 nucleotides, about 1 to 100 nucleotides, about 1 to 50 nucleotides, about 1 to 25 nucleotides, about 1 to 10 nucleotides, 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 nucleotide sequence of the encoding molecule).

[0416] According to one embodiment, the deletion comprises a deletion of at most 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, 250, 300, 350, 400, 450, or at most 500 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule).

[0417] According to certain embodiments, the deletion comprises a deletion of up to 200 nucleotides.

[0418] According to certain embodiments, the deletion comprises a deletion of up to 150 nucleotides.

[0419] According to certain embodiments, the deletion comprises a deletion of up to 100 nucleotides.

[0420] According to certain embodiments, the deletion comprises a deletion of up to 50 nucleotides.

[0421] According to certain embodiments, the deletion comprises a deletion of up to 25 nucleotides.

[0422] According to certain embodiments, the deletion comprises a deletion of up to 24 nucleotides.

[0423] According to certain embodiments, the deletion comprises a deletion of up to 23 nucleotides.

[0424] According to certain embodiments, the deletion comprises a deletion of up to 22 nucleotides.

[0425] According to certain embodiments, the deletion comprises a deletion of up to 21 nucleotides.

[0426] According to certain embodiments, the deletion comprises a deletion of up to 20 nucleotides.

[0427] According to certain embodiments, the deletion comprises a deletion of up to 15 nucleotides.

[0428] According to certain embodiments, the deletion comprises a deletion of at most 10 nucleotides.

[0429] According to certain embodiments, the deletion comprises a deletion of up to 5 nucleotides.

[0430] According to one embodiment, the modification comprises a point mutation.

[0431] According to certain embodiments, the point mutations are located (relative to the aberrantly processed transcribable RNA silencing molecule) between about 1 and 500 nucleotides, between about 1 and 250 nucleotides, between about 1 and 150 nucleotides, between about 1 and 100 nucleotides, between about 1 and 50 nucleotides, between about 1 and 25 nucleotides, between about 1 and 10 nucleotides, between about 10 and 250 nucleotides, between about 10 and 200 nucleotides, between about 10 and 150 nucleotides, between about 10 and 100 nucleotides, between about 10 and 50 nucleotides, between about 1 and 50 nucleotides, between about 1 and 10 nucleotides, between about 50 and 10 nucleotides, between about 1 and 50 nucleotides, between about 1 ... It contains point mutations of 50 nucleotides, about 50 to 100 nucleotides, or about 100 to 200 nucleotides.

[0432] According to one embodiment, the point mutation comprises a point mutation of at most 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, 250, 300, 350, 400, 450, or at most 500 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule).

[0433] According to certain embodiments, the point mutation comprises a point mutation at most 200 nucleotides.

[0434] According to certain embodiments, the point mutation comprises a point mutation at most 150 nucleotides.

[0435] According to certain embodiments, the point mutations comprise point mutations at most 100 nucleotides.

[0436] According to certain embodiments, the point mutations comprise point mutations at most 50 nucleotides.

[0437] According to certain embodiments, the point mutations comprise point mutations at a maximum of 25 nucleotides.

[0438] According to certain embodiments, the point mutations comprise point mutations at a maximum of 24 nucleotides.

[0439] According to certain embodiments, the point mutations comprise point mutations at up to 23 nucleotides.

[0440] According to certain embodiments, the point mutations comprise point mutations at a maximum of 22 nucleotides.

[0441] According to certain embodiments, the point mutations comprise point mutations at up to 21 nucleotides.

[0442] According to certain embodiments, the point mutations comprise point mutations at a maximum of 20 nucleotides.

[0443] According to certain embodiments, the point mutations comprise point mutations at a maximum of 15 nucleotides.

[0444] According to certain embodiments, the point mutations comprise point mutations in at most 10 nucleotides.

[0445] According to certain embodiments, the point mutations comprise point mutations in up to 5 nucleotides.

[0446] According to one embodiment, the modifications comprise any combination of deletions, insertions, and / or point mutations.

[0447] According to one embodiment, the modification comprises a nucleotide substitution (eg, a nucleotide exchange).

[0448] According to certain embodiments, the replacement is between about 1 and 500 nucleotides, 1 and 450 nucleotides, 1 and 400 nucleotides, 1 and 350 nucleotides, 1 and 300 nucleotides, 1 and 250 nucleotides, 1 and 200 nucleotides, 1 and 150 nucleotides, 1 and 100 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule). nucleotides, 1-90 nucleotides, 1-80 nucleotides, 1-70 nucleotides, 1-60 nucleotides, 1-50 nucleotides, 1-40 nucleotides, 1-30 nucleotides, 1-20 nucleotides, 1-10 nucleotides, 10-100 nucleotides, 10-90 nucleotides, 10-80 nucleotides, 10-70 nucleotides, 10-60 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 10-15 nucleotides, 20-30 nucleotides, 20-50 nucleotides, 20-70 nucleotides, 30-40 nucleotides, 30-50 nucleotides, 30-70 nucleotides, 40-50 nucleotides, 40-80 nucleotides, 50-60 nucleotides, 50-70 nucleotides, 50-90 nucleotides, 60-70 nucleotides, 60-80 nucleotides, 7 The replacement may be between 0 and 80 nucleotides, between 70 and 90 nucleotides, between 80 and 90 nucleotides, between 90 and 100 nucleotides, between 100 and 110 nucleotides, between 100 and 120 nucleotides, between 100 and 130 nucleotides, between 100 and 140 nucleotides, between 100 and 150 nucleotides, between 100 and 160 nucleotides, between 100 and 170 nucleotides, between 100 and 180 nucleotides, between 100 and 190 nucleotides, between 100 and 200 nucleotides, between 110 and 120 nucleotides, between 120 and 130 nucleotides, between 130 and 140 nucleotides, between 140 and 150 nucleotides, between 160 and 170 nucleotides, between 180 and 190 nucleotides, between 190 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, or between about 450 and 500 nucleotides.

[0449] According to one embodiment, the nucleotide exchange comprises nucleotide substitutions at most 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, 250, 300, 350, 400, 450, or at most 500 nucleotides (compared to the aberrantly processed transcribable RNA silencing molecule).

[0450] According to certain embodiments, the nucleotide exchange comprises nucleotide substitutions in up to 200 nucleotides.

[0451] According to certain embodiments, the nucleotide exchange comprises nucleotide substitutions in up to 150 nucleotides.

[0452] According to certain embodiments, the nucleotide exchange comprises nucleotide substitutions in up to 100 nucleotides.

[0453] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions in up to 50 nucleotides.

[0454] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions in a maximum of 25 nucleotides.

[0455] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions in up to 24 nucleotides.

[0456] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions at most 23 nucleotides.

[0457] According to certain embodiments, the nucleotide exchange is a nucleotide exchange at most 22 nucleotides. Includes leotide substitution.

[0458] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions at most 21 nucleotides.

[0459] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions in up to 20 nucleotides.

[0460] According to certain embodiments, the nucleotide exchanges comprise nucleotide substitutions in a maximum of 15 nucleotides.

[0461] According to certain embodiments, the nucleotide exchange comprises nucleotide substitutions in a maximum of 10 nucleotides.

[0462] According to certain embodiments, the nucleotide exchange comprises nucleotide substitutions at most 5 nucleotides.

[0463] According to one embodiment, when the modification is an insertion or exchange, a donor oligonucleotide is utilized (as described below).

[0464] According to one embodiment, any one or a combination of the above modifications may be performed to confer processivity of the RNA molecule into small RNAs that associate with RISC.

[0465] According to certain embodiments, gene editing (e.g., using CRISPR / Cas9 technology) in combination with donor oligonucleotides (as described below) affects altered (e.g., replaced) deletions and insertions, resulting in processivity and silencing activity of dysfunctional RNA silencing molecules. Such methods are disclosed, for example, in International Publication No. 2019 / 058255, the entire contents of which are incorporated herein by reference.

[0466] According to one embodiment, the RNA molecule is endogenous (naturally occurring, e.g., native) to the cell. It will be appreciated that the RNA molecule may also be exogenous (i.e., added from the outside and not naturally occurring within the cell) to the cell.

[0467] According to some embodiments, the RNA molecule has intrinsic translation inhibitory activity.

[0468] According to some embodiments, the RNA molecule has intrinsic RNA interference (RNAi) activity.

[0469] According to certain embodiments, the precursor nucleic acid sequence of an RNA silencing molecule (i.e., an RNAi molecule, e.g., miRNA, siRNA, piRNA, shRNA, etc.) is modified to retain its structural originality and be recognized and processed by cellular RNAi processing and execution factors.

[0470] According to certain embodiments, the precursor nucleic acid sequence of the dysfunctional RNA silencing molecule (i.e., miRNA, rRNA, tRNA, lncRNA, snoRNA, etc.) is modified to be recognized and processed by cellular RNAi processing and execution factors.

[0471] According to certain embodiments, conferring processivity to small RNAs that associate with RISC can be achieved, for example, by transforming the secondary structure of the dysfunctional RNA silencing molecule into a linear string form. This is done by restoring the structure of the dysfunctional RNA silencing molecule (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the structure of the corresponding homologous RNA silencing molecule processed into an RNA molecule associated with RISC (e.g., a wild-type precursor)), such as by translating the secondary structure into a string and comparing it to another string, such as, but not limited to, an RNAfold, using any method known in the art.

[0472] According to certain embodiments, the nucleic acid sequence of the dysfunctional RNA silencing molecule (i.e., tasiRNA, etc.) is modified to bind to factors and / or oligonucleotides (e.g., miRNA) that enable silencing activity and / or processing into silencing RNA. In a non-limiting example, the dysfunctional RNA silencing molecule is homologous to a transactivating RNA (tasiRNA) molecule, but is unable to bind to an amplifier RNA molecule, and therefore is unable to be processed into a silencing small RNA. Therefore, such an RNA silencing molecule is modified to bind to factors (e.g., amplifiers) that enable silencing activity.

[0473] According to some embodiments, the RNA-like molecule (e.g., miRNA-like) does not contain intrinsic translation inhibitory activity or intrinsic RNAi activity (i.e., the RNA-like molecule does not have intrinsic RNA silencing activity).

[0474] According to certain embodiments, when the cell is an Arabidopsis cell, the aberrantly processed transcribable nucleic acid sequences encoding RNA molecules exhibiting predetermined sequence homology ranges include those listed in Table 2 herein below.

[0475] According to certain embodiments, when the cell is a nematode cell, the aberrantly processed transcribable nucleic acid sequences encoding RNA molecules exhibiting predetermined sequence homology ranges include those listed in Table 3 herein below.

[0476] According to certain embodiments, when the cell is a human cell, the aberrantly processed transcribable nucleic acid sequences encoding RNA molecules exhibiting predetermined sequence homology ranges include those listed in Table 4 herein below.

[0477] According to one embodiment, the modification confers processibility of the RNA silencing molecule into a small RNA that binds to the first target RNA.

[0478] According to embodiments of the invention, the RNA molecule is specific for a first target RNA (e.g., a naturally occurring target RNA), does not cross-inhibit or silence the target RNA of interest unless designed to do so (as described below), and exhibits less than 100% global homology to the target gene, e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81% global homology to the target gene, as determined at the RNA or protein level by RT-PCR, Western blot, immunohistochemistry, and / or flow cytometry, sequencing, or any other detection method.

[0479] According to one embodiment, the method further comprises modifying the specificity of the RNA molecule having silencing activity in the cell (e.g., the RNA molecule to which silencing activity has been conferred), the method comprising silencing the RNA molecule toward a target RNA of interest that is different from the first target RNA. This involves introducing into a cell a DNA editing agent that redirects the silencing specificity, thereby altering the specificity of an RNA molecule that has silencing activity in that cell.

[0480] As used herein, the term " redirect silencing specificity " refers to the reprogramming of the original specificity of RNA silencing molecule to be directed to the non-natural target of RNA silencing molecule (also referred to herein as " redirection " of silencing activity).Therefore, the original specificity of RNA silencing molecule is destroyed (i.e., loss of function), and new specificity is directed to the RNA target (i.e., interest RNA) that is different from the natural target, that is, gain of function.

[0481] As used herein, the term " primary target RNA " refers to the RNA sequence that RNA silencing molecule naturally binds to.Therefore, those skilled in the art regard primary target RNA as the substrate of RNA silencing molecule (for example, be silenced by this RNA silencing molecule).

[0482] According to some embodiments, when referring to an RNAi-like molecule (e.g., an miRNA-like molecule), a first target RNA refers to an RNA sequence that would have been targeted by such an RNAi-like molecule if it had been processed in the same manner as a standard homolog of that RNAi-like molecule (e.g., the first target RNA is an RNA sequence that corresponds to the sequence that would have become the mature miRNA sequence of the miRNA-like molecule).

[0483] As used herein, the term "target RNA of interest" refers to the RNA sequence (coding or non-coding) that is to be silenced by a designed RNA silencing molecule.

[0484] As used herein, the phrase "silencing a target gene" refers to the absence or observable reduction of protein and / or mRNA products from a target gene. Thus, compared to a target gene not targeted by a designed RNA silencing molecule of the present invention, the target gene can be silenced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0485] According to one embodiment, the nucleic acid sequence of a transcribable nucleic acid sequence encoding an aberrantly processed RNA molecule that exhibits a predetermined range of sequence homology is modified to confer its ability to associate with RISC and be processed into small RNAs complementary to the target RNA of interest.

[0486] According to one embodiment, the nucleic acid sequence of the transcribable nucleic acid sequence is modified to confer a structure for an aberrantly processed RNA molecule, such that the RNA molecule associates with RISC and is processed into small RNAs that target the RNA of interest.

[0487] The results of silencing can be confirmed by examining the external characteristics of the eukaryotic cell or organism (eg, plant cell or whole plant) or by biochemical techniques (as described below).

[0488] It will be understood that the designed RNA silencing molecules of some embodiments of the present invention may have some off-target specific effect(s), so long as they do not affect the growth, differentiation, or function of eukaryotic cells or organisms, for example, by not affecting agriculturally valuable traits of plants (e.g., biomass, yield, growth, etc.).

[0489] According to one embodiment, the target RNA of interest is endogenous to the eukaryotic cell.

[0490] Exemplary endogenous target RNAs of interest in animal cells (e.g., mammalian cells) include, but are not limited to, the products of genes associated with cancer and / or apoptosis. Exemplary cancer-associated target genes include, but are not limited to, p53, BAX, PUMA, NOXA, and FAS, as described in more detail below.

[0491] Exemplary endogenous target RNAs of interest in plant cells include, but are not limited to, the products of genes that confer susceptibility to stress, infection, herbicides, or the products of genes associated with plant growth rate, crop yield, as discussed further below.

[0492] According to one embodiment, the target RNA of interest is exogenous (also referred to herein as heterologous) to the eukaryotic cell, e.g., plant cell. In such cases, the target RNA of interest is the product of a gene that is not naturally part of the genome of the eukaryotic cell (e.g., the genome of the plant).

[0493] Exemplary exogenous target RNAs in animal cells (e.g., mammalian cells) include, but are not limited to, products of genes associated with infectious diseases, such as genes of pathogens (e.g., insects, viruses, bacteria, fungi, nematodes), as discussed further herein below.

[0494] Exemplary exogenous target RNAs of interest in plant cells include, but are not limited to, the products of genes from plant pathogens, such as, but not limited to, insects, viruses, bacteria, fungi, nematodes, etc., as further discussed herein below.

[0495] The exogenous target RNA (coding or non-coding) may comprise a nucleic acid sequence that shares sequence identity with an endogenous RNA sequence of a eukaryote (e.g., a plant) (e.g., may be partially homologous to the endogenous nucleic acid sequence).

[0496] Specific binding of an RNA silencing molecule to a target RNA can be determined by computational algorithms (eg, BLAST) and verified by methods including, for example, Northern blot, in situ hybridization, QuantiGene Plex Assay, and the like.

[0497] The use of the term "complementary" or "complementary" means that an RNA silencing molecule (or at least a portion thereof present in the form of a processed small RNA, or at least one strand or portion thereof of a double-stranded polynucleotide, or a portion of a single-stranded polynucleotide) hybridizes to a target RNA or a fragment thereof under physiological conditions, causing regulation or function or repression of a target gene. For example, in some embodiments, the RNA silencing molecule inhibits 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 100 percent 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 percent sequence identity compared to a sequence of 56, 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more contiguous nucleotides.

[0498] As used herein, an RNA silencing molecule or its processed small RNA form is an RNA silencing molecule in which all nucleotides on one side of the sequence reading 5' to 3' are reversed 3' to 5'. A nucleotide sequence that is completely complementary to a reference nucleotide sequence is said to exhibit "perfect complementarity" if it is complementary to all nucleotides of the other sequence when read in reverse. A nucleotide sequence that is completely complementary to a reference nucleotide sequence exhibits a sequence identical to the reverse complementary sequence of the reference nucleotide sequence.

[0499] Methods for determining sequence complementarity are well known in the art and include, but are not limited to, bioinformatics tools well known in the art (e.g., BLAST, multiple sequence alignment).

[0500] According to one embodiment, when the RNA silencing molecule is an siRNA or is processed into an siRNA, the complementarity is in the range of 90-100% (eg, 100%) to its target sequence.

[0501] According to one embodiment, when the RNA silencing molecule is or is processed into an miRNA or piRNA, the complementarity is in the range of 33-100% to its target sequence.

[0502] According to one embodiment, when the RNA silencing molecule is an miRNA, the complementarity of the seed sequence (i.e., the 2nd to 8th nucleotides from the 5' end) to its target sequence is in the range of 85 to 100% (e.g., 100%).

[0503] According to one embodiment, the RNA silencing molecule is designed to have at least about 33%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity to the sequence of the target RNA of interest.

[0504] According to certain embodiments, the RNA silencing molecule is designed to have a minimum of 33% complementarity to the target RNA of interest (e.g., 85-100% seed match).

[0505] According to certain embodiments, the RNA silencing molecule is designed to have at least 40% complementarity to the target RNA of interest.

[0506] According to certain embodiments, the RNA silencing molecule is designed to have at least 50% complementarity to the target RNA of interest.

[0507] According to certain embodiments, the RNA silencing molecule is designed to have at least 60% complementarity to the target RNA of interest.

[0508] According to certain embodiments, the RNA silencing molecule is designed to have at least 70% complementarity to the target RNA of interest.

[0509] According to certain embodiments, the RNA silencing molecule is designed to have at least 80% complementarity to the target RNA of interest.

[0510] According to certain embodiments, the RNA silencing molecule is designed to have at least 90% complementarity to the target RNA of interest.

[0511] According to certain embodiments, the RNA silencing molecule is designed to have at least 95% complementarity to the target RNA of interest.

[0512] According to certain embodiments, the RNA silencing molecule is at least It is designed to have 96% complementarity.

[0513] According to certain embodiments, the RNA silencing molecule is designed to have at least 97% complementarity to the target RNA of interest.

[0514] According to certain embodiments, the RNA silencing molecule is designed to have at least 98% complementarity to the target RNA of interest.

[0515] According to certain embodiments, the RNA silencing molecule is designed to have at least 99% complementarity to the target RNA of interest.

[0516] According to certain embodiments, the RNA silencing molecule is designed to have 100% complementarity to the target RNA of interest.

[0517] Any of the DNA editing agents described above may be used to alter the specificity of an RNA molecule having silencing activity.

[0518] According to one embodiment, the RNA silencing molecule has a modified guide strand (silencing strand) that has about 50-100% complementarity to the target RNA of interest.

[0519] According to one embodiment, the RNA silencing molecule is modified such that the passenger strand (complementary strand) has about 50-100% complementarity to the target RNA of interest.

[0520] According to one embodiment, the RNA silencing molecule is modified so that the seed sequence (eg, in the case of an miRNA nucleotides 2-8 from the 5' end) is complementary to the target sequence.

[0521] According to one embodiment, modification of the nucleic acid sequence to confer processivity on the small RNA is performed before altering the specificity of the RNA silencing molecule.

[0522] According to one embodiment, modification of the nucleic acid sequence to confer processivity to the small RNA is performed simultaneously with modification of the specificity of the RNA silencing molecule.

[0523] According to one embodiment, the specificity of the RNA silencing molecule is altered without impairing its processability.

[0524] Thus, if the RNA silencing molecule contains non-essential structures (i.e., secondary structures of the RNA silencing molecule that do not play a role in its proper biogenesis and / or function) or is purely dsRNA (i.e., an RNA silencing molecule having complete or nearly complete dsRNA), several modifications (e.g., 20-30 nucleotides, e.g., 1-10 nucleotides, e.g., 5 nucleotides) are introduced to confer processibility and optionally modify the specificity of the RNA silencing molecule.

[0525] In another embodiment, if the RNA silencing molecule has an essential structure (i.e., the proper biogenesis and / or activity of the RNA silencing molecule is dependent on its secondary structure), larger modifications (e.g., 1 to 500 nucleotides, 10 to 250 nucleotides, 50 to 150 nucleotides, 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 confer processibility and optionally modify the specificity of the RNA silencing molecule.

[0526] According to one embodiment, a gene encoding an RNA silencing molecule is modified by replacing the sequence of the endogenous RNA silencing molecule (e.g., miRNA) with a selected RNA silencing sequence (e.g., siRNA).

[0527] According to one embodiment, the guide strand of an RNA silencing molecule, such as a miRNA precursor (pri / pre-miRNA) or a siRNA precursor (dsRNA), is modified to preserve the originality of its structure and maintain the same base-pairing profile.

[0528] According to one embodiment, the passenger strand of an RNA silencing molecule, such as a miRNA precursor (pri / pre-miRNA) or a siRNA precursor (dsRNA), is modified to preserve the originality of its structure and maintain the same base-pairing profile.

[0529] It will be appreciated that additional mutations can be introduced by additional editing events (i.e., simultaneously or sequentially).

[0530] The DNA editing agents of the present invention may be introduced into cells (e.g., eukaryotic cells) using DNA delivery methods (e.g., by expression vectors) or using DNA-free methods.

[0531] According to one embodiment, the sgRNA (or any other DNA recognition module used, depending on the DNA editing system used) may be provided to the cell as RNA.

[0532] Thus, it will be understood that the present technology relates to introducing a DNA editing agent using 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, e.g., Cas9 / gRNA ribonucleoprotein (RNP) complex transfection).

[0533] For example, Cas9 can be introduced as a DNA expression plasmid, as an in vitro transcript (i.e., RNA), or as a recombinant protein bound to an RNA moiety in a ribonucleoprotein particle (RNP). sgRNAs can be delivered, for example, as a DNA plasmid or as an in vitro transcript (i.e., RNA).

[0534] For example, microinjection (described in Cho et al., "Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins," Genetics (2013) 195:1177-1180, which is incorporated by reference herein), electroporation (described in Kim et al., "Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins," Genome Res. (2014) 24:1012-1019, which is incorporated by reference herein), or lipid-mediated transfection using, for example, liposomes (described in Zuris et al., "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo," Nat Any method known in the art for RNA or RNP transfection can be used in accordance with the present teachings, including, but not limited to, those described in [Biotechnol. (2014) doi:10.1038 / nbt.3081]. Additional methods for RNA transfection are described in their entirety in the literature. No. 20160289675, which is incorporated herein by reference.

[0535] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and vector-free. The RNA transgene can be delivered to cells as a minimal expression cassette and expressed therein without the need for any additional sequences (e.g., viral sequences).

[0536] According to one embodiment, to express an exogenous DNA editing agent of the present invention in a cell, a polynucleotide sequence encoding the DNA editing agent is ligated into a nucleic acid construct suitable for cellular expression, which includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell, either constitutively or inducibly.

[0537] Nucleic acid constructs (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences (e.g., shuttle vectors) that make the vector suitable for replication and integration in eukaryotes. Additionally, typical cloning vectors may contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. By way of example, such constructs typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or portion thereof.

[0538] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box, located 25–30 base pairs upstream of the transcription start site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. Other upstream promoter elements determine the rate at which transcription is initiated.

[0539] Preferably, the promoters utilized by the nucleic acid constructs of some embodiments of the present invention are active in the particular cell population that is transformed. Examples of cell type- and / or tissue-specific promoters include the liver-specific albumin [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphocyte-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275], in particular promoters of T cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916], or mammary gland-specific promoters such as the milk whey promoter (US Pat. No. 4,873,316 and European Patent Application Publication No. 264,166).

[0540] For expression in plant cells, the plant promoters used may be constitutive, tissue-specific, inducible, chimeric, or developmentally regulated.

[0541] Examples of preferred promoters useful in the methods of some embodiments of the present invention (in plant cells) are provided in Tables I, II, III, and IV.

[0542] [Table 1]

[0543] [Table 2]

[0544] [Table 3]

[0545] [Table 4-1] [Table 4-2]

[0546] Inducible promoters are promoters that are induced in specific plant tissues, depending on the developmental stage, or by specific stimuli such as light, temperature, chemicals, drought, high salinity, osmotic shock, stress conditions including oxidant conditions, or pathogenicity. Examples of inducible promoters include, but are not limited to, the light-inducible promoter derived from the pea rbcS gene, the promoter derived from the alfalfa rbcS gene, the promoters DRE, MYC, and MYB that are active under drought, the promoters INT, INPS, prxEa, Ha hsp17.7G4, and RD21 that are active under high salinity and osmotic stress, and the promoters hsr203J and str246C that are active under pathogenic stress.

[0547] In one embodiment, the promoter is a pathogen-inducible promoter. These promoters direct the expression of genes in plants after infection with pathogens such as bacteria, fungi, viruses, nematodes, and insects. Such promoters include those that are expressed after pathogen infection. These include those derived from pathogenicity-related proteins (PR proteins) induced by the pathogen, such as PR proteins, SAR proteins, β-1,3-glucanases, chitinases, 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.

[0548] According to one embodiment, when more than one promoter is used in an expression vector, the promoters are identical (eg, all identical, at least two identical).

[0549] According to one embodiment, when more than one promoter is used in an expression vector, the promoters are different (eg, at least two are different, all are different).

[0550] According to one embodiment, promoters in expression vectors for expression in plant cells include, but are not limited to, CaMV 35S, 2x CaMV 35S, CaMV 19S, ubiquitin, AtU626, or TaU6.

[0551] According to a particular embodiment, the promoter of the expression vector for expression in plant cells comprises the 35S promoter.

[0552] According to a particular embodiment, the promoter of the expression vector for expression in plant cells comprises the U6 promoter.

[0553] Enhancer elements can stimulate transcription from linked homologous or heterologous promoters up to 1,000-fold. Enhancers are active when placed downstream or upstream of the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the present invention include long terminal repeat sequences from various retroviruses, such as polyoma virus, human or murine cytomegalovirus (CMV); murine leukemia virus, murine or Rous sarcoma virus, and HIV. See "Enhancers and Eukaryotic Expression," Cold Spring Harbor Press, Cold Spring Harbor, NY 1983, incorporated herein by reference.

[0554] In constructing an expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural environment, although, as is known in the art, some variation in this distance can be accommodated without loss of promoter function.

[0555] Polyadenylation sequences may also be added to expression vectors to increase the efficiency of mRNA translation. Accurate and efficient polyadenylation requires two distinct sequence elements: a GU- or U-rich sequence located downstream of the polyadenylation site and a highly conserved 6-nucleotide sequence, AAUAAA, located 11 to 30 nucleotides upstream. Suitable termination and polyadenylation signals for some embodiments of the present invention include those derived from SV40.

[0556] According to a particular embodiment, the expression vector for expression in plant cells is a G7-terminated Termination sequences include, but are not limited to, termination sequences such as a termination sequence, an AtuNos termination sequence, or a CaMV-35S termination sequence.

[0557] In addition to the elements already described, expression vectors of some embodiments of the present invention may typically contain other specialized elements intended to enhance expression levels of the cloned nucleic acid or to facilitate identification of cells harboring the recombinant DNA. For example, many animal viruses contain DNA sequences that promote extra-chromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate episomally as long as the appropriate factors are provided by genes carried on the plasmid or in the genome of the host cell.

[0558] The vector may or may not contain a eukaryotic replicon. If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using an appropriate selectable marker. If the vector does not contain a eukaryotic replicon, episomal amplification is not possible. Instead, the recombinant DNA is integrated into the genome of the genetically engineered cell, where the promoter directs the expression of the desired nucleic acid.

[0559] The expression vectors of some embodiments of the present invention may further comprise additional polynucleotide sequences that allow for translation of several proteins from a single mRNA, such as, for example, an internal ribosome entry site (IRES), and sequences for integrating the promoter-chimeric polypeptide into the genome.

[0560] It will be understood that the individual elements contained within an expression vector can be arranged in a variety of configurations. For example, enhancer elements, promoters, etc., and even the polynucleotide sequence(s) encoding a DNA editing agent may be arranged in a "head-to-tail" configuration, may exist as reverse complements, or may exist as antiparallel strands in a complementary configuration. While such various configurations are likely to occur with non-coding elements of an expression vector, alternative configurations of coding sequences within an expression vector are also contemplated.

[0561] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Stratagene, pTRES available from Clontech, and derivatives thereof.

[0562] Expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses may also be used. SV40 vectors include pSVT7 and pMT2. Bovine papillomavirus-derived vectors include pBV-1MTHA, and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A, and pAV009 / A. + , pMTO10 / A + , pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, 75 inalized 75 promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0563] Viruses often contain highly specialized host defense mechanisms that have evolved to evade them. Viruses are infectious agents. Typically, viruses infect and replicate in specific cell types. The targeting specificity of viral vectors utilizes their natural specificity to specifically target a given cell type, thereby introducing a recombinant gene into the infected cell. Therefore, the type of vector used by some embodiments of the present invention depends on the cell type to be transformed. The ability to select an appropriate vector depending on the cell type to be transformed is well within the capabilities of those skilled in the art, and therefore, a general discussion of selection considerations is not provided herein. For example, bone marrow cells can be targeted using human T-cell leukemia virus type I (HTLV-I), and kidney cells can be targeted using a heterologous promoter present in the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149:51-60).

[0564] Recombinant viral vectors offer advantages such as horizontal infection and targeting specificity, making them useful for in vivo expression of DNA editing agents. Horizontal infection, for example, is inherent in the life cycle of retroviruses, in which a single infected cell produces many progeny viral particles, which then bud away from the mother cell and infect neighboring cells. This results in rapid infection of a large area, most of which were initially uninfected by the original viral particle. This contrasts with vertical infection, in which the infectious agent spreads only through daughter progeny. It is also possible to generate viral vectors that cannot spread horizontally. This feature can be useful when the desired goal is to introduce a specific gene into only a limited number of target cells.

[0565] According to one embodiment, the nucleic acid construct for expression in plant cells is a binary vector. Examples of binary vectors are 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)).

[0566] Examples of other vectors used in other methods of delivering DNA into plant cells (e.g., transfection, electroporation, bombardment, viral inoculation, etc., as discussed below) include pGE-sgRNA (Zhang et al. Nat. Comms. 2016 7:12697), pJIT163-Ubi-Cas9 (Wang et al. Nat. Biotechnol 2004 32,947-951), pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST (Belhan et al. Plant Methods 2013 11;9(1):39), pAHC25 (Christensen, A.H. & P.H. Quail, 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and / or screenable marker genes in monocotyledonous plants.Transgenic Research 5:213-218), pHBT-sGFP(S65T)-NOS (Sheen et al.Protein phosphatase activity is required for light-inducible gene expression in maize, EMBO J.12(9), 3497-3505(1993)).

[0567] According to one embodiment, to express a functional DNA editing agent, if the cleavage module (nuclease) is not an integral part of the DNA recognition unit, the expression vector may encode the cleavage module and the DNA recognition unit (e.g., sgRNA in the case of CRISPR / Cas).

[0568] Alternatively, the cleavage module (nuclease) and the DNA recognition unit (e.g., sgRNA) can be cloned into separate expression vectors. In such cases, at least two different expression vectors must be transformed into the same eukaryotic cell.

[0569] Alternatively, if nucleases are not utilized (i.e., not administered to the cells from an exogenous source), a single expression vector may be used to clone and express the DNA recognition units (e.g., sgRNAs).

[0570] According to one embodiment, the DNA editing agent comprises a nucleic acid agent encoding at least one DNA recognition unit (e.g., sgRNA) operably linked to a cis-acting regulatory element (e.g., a promoter) active in a eukaryotic cell.

[0571] 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 contain a single cis-acting regulatory element (e.g., promoter) active in eukaryotic cells to express both the nuclease and the DNA recognition unit. Alternatively, the nuclease and the DNA recognition unit may each be operably linked to a cis-acting regulatory element (e.g., promoter) active in eukaryotic cells.

[0572] According to one embodiment, the nuclease (e.g., endonuclease) and the DNA recognition unit (e.g., sgRNA) are encoded from different expression vectors, each operably linked to a cis-acting regulatory element (e.g., a promoter) active in eukaryotic cells.

[0573] According to one embodiment, the methods of some embodiments of the present invention do not include introducing a donor oligonucleotide into a cell.

[0574] According to one embodiment, the methods of some embodiments of the present invention further comprise introducing a donor oligonucleotide into the cell.

[0575] According to one embodiment, when the modification is an insertion, the method further comprises introducing a donor oligonucleotide into the cell.

[0576] According to one embodiment, if the modification is a deletion, the method further comprises introducing a donor oligonucleotide into the cell.

[0577] According to one embodiment, when the modification is a deletion and insertion (e.g., an exchange), the method further comprises introducing a donor oligonucleotide into the cell.

[0578] According to one embodiment, when the modification is a point mutation, the method further comprises introducing a donor oligonucleotide into the cell.

[0579] As used herein, the term "donor oligonucleotide" or "donor oligo" refers to an exogenous nucleotide, i.e., a nucleotide introduced externally into a cell to produce a precise change in the genome. According to one embodiment, the donor oligonucleotide is synthetic.

[0580] According to one embodiment, the donor oligo is an RNA oligo.

[0581] According to one embodiment, the donor oligo is a DNA oligo.

[0582] According to one embodiment, the donor oligo is a synthetic oligo.

[0583] According to one embodiment, the donor oligonucleotide comprises a single-stranded donor oligonucleotide (ssODN).

[0584] According to one embodiment, the donor oligonucleotide comprises a double-stranded donor oligonucleotide (dsODN).

[0585] According to one embodiment, the donor oligonucleotide comprises double-stranded DNA (dsDNA).

[0586] According to one embodiment, the donor oligonucleotide comprises a double-stranded DNA-RNA duplex (DNA-RNA duplex).

[0587] According to one embodiment, the donor oligonucleotide comprises a double-stranded DNA-RNA hybrid.

[0588] According to one embodiment, the donor oligonucleotide comprises a single-stranded DNA-RNA hybrid.

[0589] According to one embodiment, the donor oligonucleotide comprises single-stranded DNA (ssDNA).

[0590] According to one embodiment, the donor oligonucleotide comprises double-stranded RNA (dsRNA).

[0591] According to one embodiment, the donor oligonucleotide comprises single-stranded RNA (ssRNA).

[0592] According to one embodiment, the donor oligonucleotide comprises the DNA or RNA sequence for exchange (as described above).

[0593] According to one embodiment, the donor oligonucleotide is provided in a non-expression vector format or oligo.

[0594] According to one embodiment, the donor oligonucleotide comprises a DNA donor plasmid (e.g., a circular or linearized plasmid).

[0595] According to one embodiment, the donor oligonucleotide has a nucleotide sequence of about 50 to 5000, about 100 to 5000, about 250 to 5000, about 500 to 5000, about 750 to 5000, about 1000 to 5000, about 1500 to 5000, about 2000 to 5000, about 2500 to 5000, about 3000 to 5000, about 4000 to 5000, about 50 to 4000, about 100 to 4000, about 250 to 4000, about 500 to 4000, about 750-4000, approx. 1000-4000, approx. 1500-4000, approx. 2000-4000, approx. 2500-4000, approx. 3000-4000, approx. 50-3000, approx. 100-3000, approx. 250-3000, approx. 500-3000, approx. 750-3000, approx. 1000-3000, approx. 1500-3000, approx. 2000-3000, approx. 50-2000, approx. 100-2000, approx. 250-2000, approx. 500- about 2000, about 750 to 2000, about 1000 to 2000, about 1500 to 2000, about 50 to 1000, about 100 to 1000, about 250 to 1000, about 500 to 1000, about 750 to 1000, about 50 to 750, about 150 to 750, about 250 to 750, about 500 to 750, about 50 to 500, about 150 to 500, about 200 to 500, about 250 to 500, about 350 to 500, about 50 to 250, about 150 to 250, or about 200 to 250 nucleotides, and chimeric DNA-RNA hybrids.

[0596] According to certain embodiments, the donor oligonucleotide, including ssODN (eg, ssDNA or ssRNA), comprises about 200-500 nucleotides.

[0597] According to certain embodiments, the donor oligonucleotide, including a dsODN (eg, a dsDNA or a dsRNA), comprises between about 250 and 5000 nucleotides.

[0598] Exemplary donor DNAs and sgRNAs that can be used in accordance with some embodiments of the present invention are set forth in Tables 1A and 1B herein below.

[0599] According to one embodiment, to genetically replace an endogenous RNA silencing molecule (e.g., miRNA) with a selected RNA silencing sequence (e.g., siRNA), an expression vector, ssODN (e.g., ssDNA or ssRNA), or dsODN (e.g., dsDNA or dsRNA) need not be expressed in the cell but can serve as a non-expressing template. According to certain embodiments, in such cases, only the DNA editing agent (e.g., Cas9 / sgRNA module) needs to be expressed when provided in DNA form.

[0600] According to some embodiments, a DNA editing agent (e.g., a gRNA) may be introduced into a eukaryotic cell with or without the use of an oligonucleotide donor DNA or RNA (e.g., as discussed herein) to gene-edit an endogenous RNA silencing molecule without the use of nucleases.

[0601] According to one embodiment, the donor oligonucleotide is introduced into the cell using any of the methods described above (eg, using an expression vector or RNP transfection).

[0602] According to one embodiment, the sgRNA and DNA donor oligonucleotide are co-introduced into a cell (e.g., a eukaryotic cell). It will be understood that any additional factors (e.g., nucleases) may also be co-introduced thereto.

[0603] According to one embodiment, the sgRNA and DNA donor oligonucleotide are co-introduced into the plant cell (e.g., via bombardment gun). It will be understood that any additional factors (e.g., nucleases) may also be co-introduced.

[0604] According to one embodiment, the sgRNA is introduced into the cell before (e.g., within minutes or hours of) the DNA donor oligonucleotide. It will be understood that any additional factors (e.g., nucleases) may be introduced before, simultaneously with, or after the sgRNA or DNA donor oligonucleotide.

[0605] According to one embodiment, the sgRNA is introduced into the cell after (e.g., within minutes or hours of) the DNA donor oligonucleotide. It will be understood that any additional factors (e.g., nucleases) may be introduced before, simultaneously with, or after the sgRNA or DNA donor oligonucleotide.

[0606] According to one embodiment, a composition for genome editing is provided comprising at least one sgRNA and a DNA donor oligonucleotide.

[0607] According to one embodiment, a composition for genome editing is provided comprising at least one sgRNA, a nuclease (e.g., an endonuclease), and a DNA donor oligonucleotide.

[0608] According to one embodiment, at least one sgRNA is operably linked to a plant-expressible promoter.

[0609] The DNA editing agents and optionally donor oligos of some embodiments of the present invention may be administered to a single cell, to a group of cells (e.g., the plant cells, primary cells, or cell lines described above), or to an organism (e.g., the plants, mammals, birds, fish, and insects described above).

[0610] A variety of methods can be used to introduce the expression vectors or donor oligos of some embodiments of the invention into eukaryotic cells (e.g., stem cells or plant cells). Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press. Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, microinjection, particle bombardment, and infection with recombinant viral vectors. Furthermore, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0611] Thus, delivery of nucleic acids can be achieved by transformation of protoplasts (see, e.g., U.S. Pat. No. 5,508,184); by desiccation / inhibition-mediated DNA uptake (see, e.g., Potrykus et al., J. Immunol. 2004; 2006; 2007; 2009; 2010). al. (1985) Mol. Gen. Genet. 199:183-8); by electroporation (see, e.g., U.S. Pat. No. 5,384,253); by agitation with silicon carbide fibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and 6,384,301); DNA The antibodies may be introduced into cells in embodiments of the present invention by any method known to those skilled in the art, including, but not limited to, by acceleration of particles coated with the antibody (see, e.g., 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 nanoparticles, nanocarriers, and cell-penetrating peptides (see, e.g., WO 201126644A2, WO 2009046384A1, WO 2008148223A1). .

[0612] Other methods of transfection include transfection reagents (e.g., Lipofectin, ThermoFisher), dendrimers (Kukowska-Latallo, JF et al., 1996, Proc. Natl. Acad. Sci. USA 93, 4897-902), and cell-penetrating peptides (Mae et al., 2005,Internalization of cell-penetrating Examples include the use of peptides into tobacco protoplasts, Biochimica et Biophysica Acta 1669(2):101-7, or polyamines (Zhang and Vinogradov, 2010, Short biodegradable polyamines for gene delivery and transfection of brain capillary endothelial cells, J Control Release, 143(3):359-366).

[0613] According to certain embodiments, for introducing DNA into cells (e.g., plant cells, e.g., protoplasts), the method involves 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.

[0614] Introduction of nucleic acids into cells (e.g., eukaryotic cells) by viral infection offers several advantages over other methods, such as lipofection and electroporation, because higher transfection efficiencies can be obtained due to the infectivity of viruses.

[0615] Currently, preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex virus I, and adeno-associated virus (AAV), and lipid-based systems. Lipids useful for lipid-mediated gene transfer include DOTMA, DOPE, and DC-Chol (Tonkinson et al., Cancer Investigation, 14(1):54-65(1996)). For gene therapy, preferred constructs are viruses, most preferably adenovirus, AAV, lentivirus, or retrovirus. Viral constructs, such as retroviral constructs, contain at least one transcription promoter / enhancer or locus-defining element(s), or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA export, or post-translational modification of messengers. Such vector constructs also include a packaging signal, long terminal repeat (LTR) or a portion thereof, and plus-strand and minus-strand primer binding sites appropriate for the virus used, unless already present in the viral construct. Furthermore, such constructs typically include a signal sequence for secretion of the peptide from the host cell in which it is placed. Preferably, the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of a polypeptide variant of some embodiments of the present invention. Optionally, the construct may include a signal directing polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other non-viral vectors, such as cationic lipids, polylysine, and dendrimers, may also be used. The inserted In addition to containing the elements necessary for the transcription and translation of the expressed coding sequence, the expression constructs of some embodiments of the present invention may also contain sequences engineered to enhance the stability, production, purification, yield, or toxicity of the expressed peptide.

[0616] In certain embodiments, bombardment is used to introduce foreign genes into eukaryotic cells (e.g., non-plant cells, e.g., animal cells, e.g., mammalian cells). In one embodiment, the method is transient. Bombardment of eukaryotic cells (e.g., mammalian cells) is also taught in Uchida M et al., Biochim Biophys Acta. (2009) 1790(8):754-64, which is incorporated herein by reference.

[0617] According to one embodiment, plant cells can be stably or transiently transformed with the nucleic acid constructs of some embodiments of the present invention. In stable transformation, the nucleic acid molecules of some embodiments of the present invention are integrated into the plant genome, thereby representing a stable and heritable trait. In transient transformation, the nucleic acid molecules are expressed in the transformed cells but are not integrated into the genome, thereby representing a transient trait.

[0618] Various methods exist for introducing foreign genes into both 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).

[0619] The basic 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, eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, CJ, Butterworth. Publishers, Boston, Mass. (1989) p.93-112. (ii) Direct uptake of DNA, including: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) pp. 52-68; direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074; DNA uptake induced by brief electric shock to 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 by particle bombardment, Klein et al. al.Bio / Technology(1988)6:559-563;McCabe et al. al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by using a micropipette system: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenbe rg, Physiol. Plant. (1990) 79:213-217; glass fiber or silicon carbide whisker transformation of cell cultures, embryos, or callus tissue, U.S. Patent No. 5,464,765, or by direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, GPand Mantell, SHand Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0620] The Agrobacterium system involves the use of a plasmid vector containing a defined DNA segment that is integrated into the plant's genomic DNA. Methods for inoculating plant tissue vary depending on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure, which can be performed with any tissue explant, providing an excellent source for initiating whole plant differentiation. Horsch et al., Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) pp. 1-9. An adjunct approach uses the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is particularly viable for creating transgenic dicotyledonous plants.

[0621] According to one embodiment, an Agrobacterium-free expression method is used to introduce the foreign gene into the plant cell. According to one embodiment, the Agrobacterium-free expression method is transient. According to a particular embodiment, a bombardment method is used to introduce the foreign gene into the plant cell. According to another particular embodiment, plant root bombardment is used to introduce the foreign gene into the plant cell. Exemplary bombardment methods that can be used according to some embodiments of the present invention are discussed in the Examples section below.

[0622] Additionally, various cloning kits or gene synthesis can be used in accordance with the teachings of some embodiments of the present invention.

[0623] After stable transformation, plants are propagated. The most common method of plant propagation is by seed. However, regeneration by seed propagation has the problem of heterozygosity, which is a loss of crop uniformity because plants produce seeds according to genetic variance governed by Mendelian laws. Essentially, each seed is genetically different and grows with unique traits. Therefore, it is preferable to generate transformed plants so that the regenerated plants have the same traits and characteristics as the parent transgenic plant. Therefore, it is preferable to regenerate transformed plants by micropropagation, which allows for rapid and consistent regeneration of genetically identical transformed plants.

[0624] Micropropagation is the process of growing a new generation of plants from a single piece of tissue cut from a selected parent plant or variety. This process allows for the reproduction of large quantities of plants with desired traits. The newly generated plants are genetically identical to the original plant and possess all of the characteristics of the original plant. Micropropagation (or cloning) can produce large quantities of high-quality plant material in a short period of time, allowing for the rapid propagation of selected varieties while retaining the characteristics of the original transgenic or transformed plant. The advantages of clonal plants are the speed of plant propagation and the quality and uniformity of the plants generated.

[0625] Micropropagation is a multi-step procedure that requires changing culture media or growth conditions between steps. Thus, the micropropagation process involves four basic steps: step 1, initial tissue culture; step 2, tissue culture propagation; step 3, differentiation and plant formation; and step 4, greenhouse cultivation and hardening. During step 1, initial tissue culture, the tissue culture is established and certified free of contaminants. During stage 2, the initial tissue culture is propagated until a sufficient number of tissue samples are generated to meet production goals. During stage 3, the tissue samples grown in stage 2 are divided and grown into individual plantlets. In stage 4, the transformed plantlets are transferred to a greenhouse for hardening, where the plants' tolerance to light is gradually increased so that they can grow in their natural environment.

[0626] Although stable transformation is currently preferred, transient transformation of leaf cells, meristematic cells, or whole plants is also contemplated by some embodiments of the present invention.

[0627] Transient transformation can be achieved by any of the direct DNA transfer methods described above or by viral infection with modified plant viruses.

[0628] Viruses that have been shown to be useful for transforming plant hosts include CaMV, TMV, TRV, and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), European Patent No. 67,553 (TMV), Japanese Patent Application Laid-Open No. 63-14693 (TMV), European Patent No. 194,809 (BV), European Patent No. 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 use in expressing foreign DNA in many hosts, including plants, are described in International Publication No. WO 87 / 06261.

[0629] In addition to the above references, the construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants is also described by 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.

[0630] If the virus is a DNA virus, suitable modifications may be made to the virus itself. Alternatively, to facilitate the construction of a desired viral vector carrying foreign DNA, the virus may first be cloned into a bacterial plasmid. The virus may then be excised from the plasmid. If the virus is a DNA virus, the viral DNA may be linked to a bacterial replication origin and then replicated by the bacteria. Transcription and translation of this DNA produces a coat protein that encapsidates the viral DNA. If the virus is an RNA virus, the virus is generally cloned as cDNA and inserted into a plasmid. The plasmid is then used for all constructions. The viral sequence in the plasmid is then transcribed, and the viral genes are translated to produce a coat protein(s) that encapsidate the viral RNA, thereby producing an RNA virus.

[0631] In addition to the above references, construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants, such as those contained in the constructs of some embodiments of the present invention, is taught by U.S. Pat. No. 5,316,931.

[0632] In one embodiment, the coding sequence for the native coat protein is deleted from the viral nucleic acid, and a coding sequence for a non-native plant viral coat protein and a non-native promoter, preferably a non-native promoter, is used that is capable of being expressed in the plant host, packaging the recombinant plant viral nucleic acid, and ensuring systemic infection of the host with the recombinant plant viral nucleic acid. Plant viral nucleic acids are provided in which a subgenomic promoter for a native coat protein coding sequence has been inserted. Alternatively, the coat protein gene may be inactivated by inserting a non-native nucleic acid sequence therein so that the protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing an adjacent gene or nucleic acid sequence in the plant host and is incapable of recombining with each other and with the native subgenomic promoter. When more than one nucleic acid sequence is included, the non-native (foreign) nucleic acid sequence may be inserted adjacent to the native plant viral subgenomic promoter or the native and non-native plant viral subgenomic promoters. The non-native nucleic acid sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product.

[0633] In a second embodiment, a recombinant plant viral nucleic acid is provided similar to the first embodiment, except that instead of the coding sequence for the non-native coat protein, the coding sequence for the native coat protein is positioned adjacent to one of the subgenomic promoters for the non-native coat protein.

[0634] In a third embodiment, a recombinant plant viral nucleic acid is provided in which a native coat protein gene is adjacent to its subgenomic promoter and one or more non-native subgenomic promoters are inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombining with each other and with the native subgenomic promoters. A non-native nucleic acid sequence may be inserted adjacent to the non-native subgenomic plant viral promoter such that the sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce a desired product.

[0635] In a fourth embodiment, a recombinant plant viral nucleic acid is provided similar to the third embodiment, except that the coding sequence for the native coat protein is replaced with a coding sequence for a non-native coat protein.

[0636] The viral vector is encapsidated by a coat protein encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect a suitable host plant. The recombinant plant viral nucleic acid is capable of replicating in the host, spreading systemically in the host, and transcribing or expressing the foreign gene(s) (isolated nucleic acid) in the host to produce the desired protein.

[0637] In addition to the above, the nucleic acid molecules of some embodiments of the present invention can also be introduced into the chloroplast genome, thereby allowing chloroplast expression.

[0638] A technique for introducing an exogenous nucleic acid sequence into the genome of a chloroplast is known. This technique involves the following steps: First, plant cells are chemically treated to reduce the number of chloroplasts to approximately one per cell. Then, an exogenous nucleic acid is introduced into the cells via particle bombardment, with the goal of introducing at least one exogenous nucleic acid molecule into the chloroplast. The exogenous nucleic acid is selected so that it can be integrated into the chloroplast genome via homologous recombination, facilitated by enzymes native to chloroplasts. For this purpose, the exogenous nucleic acid contains, in addition to the gene of interest, at least one nucleic acid stretch derived from the chloroplast genome. Furthermore, the exogenous nucleic acid contains a selectable marker that aids in such a selection procedure to ensure that all or substantially all copies of the chloroplast genome after sequential selection contain the exogenous nucleic acid. For further details regarding this technique, see U.S. Pat. No. 4,945,050, incorporated herein by reference. and 5,693,507. In this manner, polypeptides can be produced by the chloroplast protein expression system and incorporated into the inner membrane of the chloroplast.

[0639] Regardless of the transformation / infection method used, the present teachings further select for transformed cells that contain genome editing events.

[0640] According to certain embodiments, selection is performed such that only cells that have successfully made the correct modification (e.g., replacement, insertion, deletion, point mutation) at a particular locus are selected, and thus cells containing any event involving a modification (e.g., insertion, deletion, point mutation) at an unintended locus are not selected.

[0641] According to one embodiment, selection of modified cells can be performed at the phenotypic level, by detection of a molecular event, by detection of a fluorescent reporter, or by growth in the presence of selection (e.g., resistance to an antibiotic or other selectable marker, e.g., a drug in the case of TP53 silencing, i.e., Nutlin3).

[0642] According to one embodiment, the selection of modified cells is performed by analyzing the biogenesis and occurrence of newly edited RNA silencing molecules (e.g., the presence of newly edited miRNAs, siRNAs, piRNAs, tasiRNAs, etc.).

[0643] According to one embodiment, the selection of modified cells is performed by analyzing the silencing activity and / or specificity of the RNA silencing molecule or its processed small RNA forms for the target RNA of interest, to determine at least one eukaryotic cell or organism phenotype of the organism encoding the target RNA of interest, such as cell size, growth rate / inhibition, cell shape, cell membrane integrity, tumor size, tumor shape, coloration of the organism, size of the organism, infection parameters in the organism (e.g., viral load or bacterial load), or inflammation parameters in the organism (e.g., fever or redness), coloration of plant leaves, e.g., partial or complete chlorophyll expression in leaves and other organs, or the like. This can be done by examining the presence or absence of complete loss of flower buds (chlorosis), necrotic patterns, flower coloration, fruit traits (e.g., shelf life, firmness, and aroma), growth rate, plant size (e.g., dwarfism), crop yield, biotic stress resistance (e.g., disease resistance, nematode mortality, beetle oviposition rate, or other resistance phenotypes associated with bacteria, viruses, fungi, parasites, insects, weeds, and either cultivated or native plants), crop yield, metabolic profile, fruit traits, biotic stress resistance, abiotic stress resistance (e.g., heat / cold tolerance, drought tolerance, salt tolerance, resistance to allyl alcohol, or resistance to nutrient deficiencies such as phosphorus (P)).

[0644] According to one embodiment, the silencing specificity of an RNA silencing molecule is determined genotypically, such as by the expression or lack of expression of a gene.

[0645] According to one embodiment, the silencing specificity of an RNA silencing molecule is determined phenotypically.

[0646] According to one embodiment, the phenotype of the eukaryotic cell or organism is determined before the genotype.

[0647] According to one embodiment, the genotype of the eukaryotic cell or organism is determined before the phenotype.

[0648] According to one embodiment, the selection of modified cells is performed by analyzing the silencing activity and / or specificity of the RNA silencing molecule for the target RNA of interest by measuring the RNA levels of the target RNA of interest, for example, by Northern blotting, nuclease protection assays, in situ hybridization, etc. This can be done using any method known in the art, such as quantitative RT-PCR, or immunoblotting.

[0649] According to one embodiment, the selection of modified cells is carried out by analyzing eukaryotic cells or clones containing DNA editing events, also referred to herein as "mutations" or "edits," depending on the type of editing desired, such as insertions, deletions, insertion-deletions (indels), inversions, substitutions, and combinations thereof.

[0650] Methods for detecting sequence variations are well known in the art and include, but are not limited to, DNA and RNA sequencing (e.g., next-generation sequencing), electrophoresis, enzyme-based mismatch detection assays, and hybridization assays such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot, Northern blot, and dot blot analysis. Various methods used to detect single nucleotide polymorphisms (SNPs) may also be used, such as PCR-based T7 endonuclease, heteroduplex, and Sanger sequencing, or PCR followed by restriction enzyme digestion to detect the appearance or disappearance of unique restriction site(s).

[0651] Another method for verifying the presence of DNA editing events such as indels involves mismatch cleavage assays, which utilize structure-selective enzymes (e.g., endonucleases) that recognize and cleave mismatched DNA.

[0652] According to one embodiment, selection of transformed cells is performed by flow cytometry (FACS) selection of transformed cells that exhibit fluorescence emitted by the fluorescent reporter. After FACS sorting, the positively selected pool of transformed eukaryotic cells displaying the fluorescent marker can be collected and an aliquot can be used to test for DNA editing events as described above.

[0653] If an antibiotic selection marker is used, after transformation, the eukaryotic cells are cultured in the presence of selection (e.g., an antibiotic), e.g., in cell culture, or until the plant cells develop into colonies, i.e., clones, and microcallus. A portion of the cells in the cell culture or of the callus cells are then analyzed (verified) for DNA editing events as described above.

[0654] According to one embodiment of the present invention, the method further comprises verifying the complementarity of the endogenous RNA silencing molecule to the target RNA of interest in the transformed cell.

[0655] As described above, after modification of the gene encoding the RNA silencing molecule, the RNA silencing molecule 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 to the sequence of the target RNA of interest.

[0656] Specific binding of the designed RNA silencing molecule or its processed small RNA form to the target RNA of interest can be determined by any method known in the art, for example, by computational algorithms (e.g., BLAST), and verified by methods including, for example, Northern blot, in situ hybridization, QuantiGene Plex Assay, etc.

[0657] It will be understood that a positive eukaryotic cell or clone (e.g., a plant cell clone) can be homozygous or heterozygous for the DNA editing event. In the case of a heterozygous cell, the cell (e.g., in the case of a diploid plant cell) contains an RNA silencing molecule. It may contain copies of the modified gene and copies of the unmodified gene. A person skilled in the art will select cells for further culture / regeneration depending on the intended use.

[0658] According to one embodiment, if a transient method is desired, eukaryotic cells or clones (e.g., plant cell clones) exhibiting the presence of a DNA editing event are optionally further analyzed to select for the presence of a DNA editing agent, i.e., the loss of a DNA sequence encoding the DNA editing agent. This can be done by analyzing the loss of expression of the DNA editing agent (e.g., at the mRNA or protein level), for example, by fluorescent detection of GFP or q-PCR, HPLC, etc.

[0659] According to one embodiment, if a transient method is desired, eukaryotic cells or clones (e.g., plant cell clones) may be analyzed for the presence of a nucleic acid construct described herein or a portion thereof, e.g., a nucleic acid sequence encoding a DNA editing agent. This can be confirmed by fluorescence microscopy, q-PCR, FACS, and / or any other method, such as Southern blot, PCR, sequencing, HPLC, etc.

[0660] Positive eukaryotic cell clones can be preserved (eg, cryopreserved).

[0661] Alternatively, the eukaryotic cells may be further cultured, eg, maintained in an undifferentiated state for extended periods of time, and induced to differentiate into other cell types, tissues, organs, or organisms as desired.

[0662] According to one embodiment, when the eukaryote is a plant, the plant is crossed to obtain a plant that does not contain a DNA editing agent (e.g., an endonuclease), as described below.

[0663] Alternatively, plant tissue culture techniques can be used to regenerate whole plants from plant cells (e.g., protoplasts) by first growing them into clusters of plant cells, causing them to develop into callus, and then regenerating shoots from the callus (callus development). To grow the protoplasts into callus and regenerate shoots, the tissue culture medium must contain the appropriate balance of plant growth regulators, which must be customized for each plant species.

[0664] Protoplasts can also be used in plant breeding using a technique called protoplast fusion. Fusion of protoplasts from different species is induced using an electric field or a solution of polyethylene glycol. This technique can be used to generate somatic cell hybrids in tissue culture.

[0665] Methods for regenerating protoplasts are well known in the art. Several factors influence protoplast isolation, culture, and regeneration: genotype, donor tissue and its pretreatment, enzymatic treatment to isolate protoplasts, protoplast culture method, culture medium, and physical environment. For a complete review, see Maheshwari et al. 1986 Differentiation of Protoplasts and of Transformed Plant Cells: 3-36. Springer-Verlag, Berlin.

[0666] The regenerated plants can be subjected to further breeding and selection as deemed appropriate by those skilled in the art.

[0667] Accordingly, embodiments of the present invention further relate to plants, plant cells, and plant processing products comprising RNA silencing molecules capable of silencing a target RNA of interest produced according to the present teachings.

[0668] According to one aspect of the present invention, there is provided a method of generating a plant with reduced expression of a target gene, the method comprising: (a) breeding a plant of some embodiments of the present invention; and (b) selecting progeny plants with reduced expression of a target RNA of interest, or progeny that comprise a silencing RNA molecule specific for the target RNA of interest and that do not contain a DNA editing agent, thereby generating a plant with reduced expression of the target gene.

[0669] According to one aspect of the present invention, there is provided a method for generating a plant comprising an RNA molecule having silencing activity against a target RNA of interest, comprising the steps of: (a) breeding a plant of some embodiments of the present invention; (b) selecting progeny plants that comprise the RNA molecule having silencing activity against the target RNA of interest, or progeny that comprise a silencing specificity in the RNA molecule for the target RNA of interest and that do not contain the DNA editing agent, thereby generating plants that comprise the RNA molecule having silencing activity against the target RNA of interest.

[0670] According to one aspect of the invention, there is provided a method of producing a plant or plant cell of some embodiments of the invention, comprising growing the plant or plant cell under conditions that allow propagation.

[0671] The term "plant" as used herein includes whole plants, grafted plants, plant ancestors and progeny, and plant parts including seeds, shoots, stems, roots (including tubers), rootstocks, shoots, and plant cells, tissues, and organs. Plants may be in any form, including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that may be useful in the methods of the present invention include all plants belonging to the superfamily Chlorophyta, in particular monocotyledonous and dicotyledonous plants, including forage or fodder plants, ornamental plants, food crops, trees, or shrubs selected from the list comprising: Acacia species, Acer species, Actinidia species, Aesculus species, Agathis australis, Albizia amara, Alsophila tricolor, Andropogon species, Arachis species, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga plurijuga, Birch (Betuila) species, Brassica species, Bruguiera gymnorrhiza, Burkea 90inalize, Butea frondosa, Cadaba 90inalize, Calliandra species, Tea plant (Camellia sinensis), Cannabaceae, Canna indica, Cannabis species, Cannabis sativa, Cannabis sativa, Industrial hemp, Capsicum species, Cassia species, Centroema pubescens, Chacoomeles species, Cinnamomum cassia, Coffee arabica, Colophospermum mopane, Coronillia varia, Cotoneaster 90inalize, Crataegus species, Cucumis species, Cypress species, Silver fern, Quince, Cryptomeria japonica japonica), Cymbopogon species, Cynthea dealbata ), Quince, Veronica (Dalbergia monetaria), Davallia 90inalized90, Desmodium species, Dicksonia squarosa, Dibeteropogon amplectens, Dioclea species, Dolichos species, Dorycnium rectum, Echinochloa pyramidalis, Ehraffia species, Finger millet (Eleusine coracana, Eragrestis species, Erythrina species, Eucalypfus species, Eucalyptus species, Euclea schimperi, Eulalia vi / losa, Pagopyrum species, Feijoa sellowlana, Fragaria species, Flemingia species, Freycinetia banksli, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia species, Gossypium hirsutum, Grevillea species, Guibourtia coleosperma, Hedysarum species, Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyperrhenia rufa, Hypericum erectum, Hyperifera dissolute, Indigo incamataincamata, Iris species, Leptarrhena pyrolifolia, Lespediza species, Lettuca species, Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus species, Macrotyloma axillare, Malus species, Cassava (Manihot esculenta), Medicago saliva, Metasequoia glyptostroboides, Musa sapientum sapientum, banana, Nicotianum species, Onobrychis species, Ornithopus species, Oryza species, Peltophorum africanum, Pennisetum species, avocado (Persea gratissima), petunia species, Phaseolus species, Canary palm (Phoenix canariensis), Phormium cookianum, Photinia species, Picea glauca, Pinus species, pea (Pisum sativam), Podocarpus totara totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus species, Prosopis cineraria, Douglas fir (Pseudotsuga menziesii), Pterolobium stellatum, Pyrus communis, Quercus species, Rhaphiolepsis 90 inalized90inalized), Rhopalostilis sapida ( Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes species, Robinia pseudoacacia, Rosa species, Rubus species, Salix species, Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia species, Sporobolus fimbriatus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Polygonum species, Bald cypress (Taxodium distichum), Themeda triandra, Trifolium species, Triticum species, Western hemlock (Tsuga heterophylla), Vaccinium species, Vicia species, Vitis vinifera, Watsonia pyramidata, Calla lily (Zantedeschia aethiopica), Corn (Zea mays), Amaranth, Artichoke, Asparagus, Broccoli, Brussels sprouts sprouts), cabbage, canola, carrots, cauliflower, celery, collard greens, flax, kale, lentils, rapeseed, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugarcane, sunflower, tomato, pumpkin, tea plant. Alternatively, algae and other non-green plants can be used in the methods of some embodiments of the present invention.

[0672] According to certain embodiments, the plant is a crop, a flowering plant, or a tree.

[0673] According to certain embodiments, the plant is a woody plant species, such as Actinidia chinensis (Actinidiaceae), Manihotesculenta (Euphorbiaceae), Firiodendron tulipera (Magnoliaceae), Populus (Salicaceae), Santalum album (Santalaceae), elm (Ulmus) (Ulmaceae), and different species of the families Rosaceae (Malus, Prunus, Pyrus) and Rutaceae (Citrus, Microcitrus), gymnosperms, for example Picea abies glauca and loblolly pine (Pinus taeda), forest trees (e.g., birch (Betulaceae), beech (Fagaceae), gymnosperms, and tropical tree species), fruit trees, shrubs or herbs, such as (banana, cocoa, coconut palm, coffee, date palm, grape, and tea), and oil palm.

[0674] According to particular embodiments, the plant is a tropical crop, such as coffee, macadamia, banana, pineapple, taro, papaya, mango, barley, beans, cassava, chickpea, cacao (chocolate), cowpea, maize (corn), millet, rice, sorghum, sugarcane, sweet potato, tobacco, taro, tea, or yam.

[0675] "Grain," "seed," or "bean" refers to the reproductive unit of a flowering plant that can grow into another flowering plant. As used herein, these terms are used synonymously and interchangeably. do.

[0676] According to certain embodiments, the plant is a plant cell, for example a plant cell in an embryonic cell suspension.

[0677] According to certain embodiments, the plant comprises plant cells produced by the methods of some embodiments of the present invention.

[0678] According to one embodiment, breeding comprises crossing or selfing.

[0679] The term "crossing," as used herein, refers to the fertilization of a female plant (or gamete) by a male plant (or gamete). The term "gamete" refers to a haploid reproductive cell (egg or sperm) produced in a plant by mitosis from a gametophyte and involved in sexual reproduction, in which two gametes of the opposite sex fuse to form a diploid zygote. The term generally includes reference to pollen (including sperm cells) and ovules (including egg cells). Thus, "crossing" generally refers to the fertilization of an ovule from one individual with pollen from another individual, while "selfing" refers to the fertilization of an ovule from one individual with pollen from the same individual. Crossing is widely used in plant breeding, where one chromosome from the mother crosses with one chromosome from the father, resulting in the mixing of genomic information between the two plants. This results in new combinations of genetically inherited traits.

[0680] As mentioned above, plants may be crossed to obtain plants that are free of undesirable factors, such as DNA editing agents (e.g., endonucleases).

[0681] According to some embodiments of the invention, the plant is non-transgenic.

[0682] According to some embodiments of the invention, the plant is a transgenic plant.

[0683] According to one embodiment, the plant is a non-genetically modified (non-GMO) plant.

[0684] According to one embodiment, the plant is a genetically modified (GMO) plant.

[0685] According to one embodiment, there is provided a seed of a plant produced according to the methods of some embodiments of the present invention.

[0686] According to one embodiment, there is provided a method for producing plants with increased stress tolerance, increased yield, increased growth rate, or improved yield quality, the method comprising: (a) breeding a plant of some embodiments of the invention; and (b) selecting progeny plants with increased stress tolerance, increased yield, increased growth rate, or improved yield quality.

[0687] The phrase "stress tolerance," as used herein, refers to the ability of a plant to withstand biotic or abiotic stress without substantial alterations in metabolism, growth, productivity, and / or survival rate.

[0688] The phrase "abiotic stress," as used herein, refers to exposure of a plant, plant cell, etc. to a non-living ("abiotic") physical or chemical agent that adversely affects the metabolism, growth, development, reproduction, or survival (collectively, "growth") of the plant. For example, water (e.g., flood, drought, or dehydration), anaerobic conditions (e.g., lower oxygen or high CO2), abnormal osmotic conditions (e.g., osmotic stress), salinity, or temperature (e.g., high temperature / heat, low temperature, Plants can be exposed to abiotic stress due to environmental factors such as freezing or frost, exposure to pollutants (e.g., heavy metal toxicity), anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or nitrogen limitation), air pollution, or ultraviolet radiation.

[0689] The phrase "biotic stress," as used herein, refers to exposure of plants, plant cells, etc. to living ("biotic") organisms that adversely affect the metabolism, growth, development, reproduction, or survival (collectively, "growth") of the plant. Biotic stress can be caused, for example, by bacteria, viruses, fungi, parasites, beneficial and pest organisms, weeds, and cultivated or native plants.

[0690] The phrase "yield" or "plant yield," as used herein, refers to increased plant growth (growth rate), increased crop growth, increased biomass, and / or increased production of plant products (including grains, fruits, seeds, etc.).

[0691] According to one embodiment, to generate plants with increased stress tolerance, increased yield, increased growth rate, or improved yield quality, RNA silencing molecules are designed to target an RNA of interest in a gene of the plant that confers susceptibility to stress, reduced yield, slowed growth rate, or reduced yield quality.

[0692] According to one embodiment, exemplary susceptible plant genes that may be targeted (e.g., knocked out) include, but are not limited to, susceptible S genes, such as those present in the locus known as MLO (downy mildew locus O).

[0693] According to one embodiment, a plant produced by the method comprises at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% increase in stress tolerance, increased yield, improved yield quality, or increased growth rate compared to a plant not produced by the method.

[0694] Any method known in the art for assessing increased stress tolerance can be used in accordance with the present invention. Exemplary methods for assessing increased stress tolerance include, but are not limited to, down-regulating PagSAP1 in poplar to increase salt stress tolerance as described in Yoon, SK., Bae, EK., Lee, H. et al. Trees (2018) 32: 823. www (dot) doi (dot) org / 10.1007 / s00468-018-1675-2) and down-regulating SlbZIP38 to increase drought tolerance in tomato (Pan Y et al. Genes 2017, 8, 402; doi: 10.3390 / genes8120402, which is incorporated herein by reference).

[0695] Any method known in the art for assessing yield increase can be used in accordance with the present invention. Exemplary methods for assessing yield increase include, but are not limited to, reducing DST expression in rice as described in Ar-Rafi Md. Faisal, et al., AJPS Vol. 8 No. 9, August 2017 DOI: 10.4236 / ajps.2017.89149; and down-regulating BnFTA in canola to increase yield as described in Wang Y et al., Mol Plant. 2009 Jan; 2(1): 191-200. doi: 10.1093 / mp / ssn088), both of which are incorporated herein by reference.

[0696] Any method known in the art for assessing an increase in growth rate can be used in accordance with the present invention. Exemplary methods for assessing an increase in growth rate are described in reference Examples of methods include, but are not limited to, reducing the expression of BIG BROTHER or GA2-OXIDASE in Arabidopsis thaliana to improve growth and biomass as described in Marcelo de Freitas Lima et al. Biotechnology Research and Innovation (2017) 1, 14---25, which is incorporated herein by reference.

[0697] Any method known in the art for assessing improved yield quality can be used in accordance with the present invention. Exemplary methods for assessing improved yield quality include, but are not limited to, downregulation of OsCKX2 in rice, which results in more tillers, more grain production, and heavier grains, as described in Yeh S_Y et al. Rice (NY). 2015; 8:36; and reduction of OMT levels in many plants, which alters lignin accumulation and increases the absorption rate of substances for industrial purposes, as described in Verma SR and Dwivedi UN, South African Journal of Botany Volume 91, March 2014, Pages 107-125, both of which are incorporated herein by reference.

[0698] According to one embodiment, the method further allows for the production of plants comprising increased sweetness, increased sugar content, increased aroma, improved ripening control, increased water stress tolerance, increased heat stress tolerance, and increased salt tolerance. One of skill in the art will understand how to select target RNA sequences for modification using the methods described herein.

[0699] According to one embodiment, there is provided a method for producing a plant that is tolerant or resistant to a pathogen or pest, the method comprising: (a) breeding a plant of some embodiments of the present invention; and (b) selecting a progeny plant that is tolerant or resistant to the pathogen or pest.

[0700] According to one embodiment, the target RNA of interest is of a gene in a plant that confers susceptibility to a pathogen or pest.

[0701] According to one embodiment, the target RNA of interest is that of a gene of a pathogen.

[0702] According to one embodiment, the target RNA of interest is that of a gene of a pest organism.

[0703] As used herein, the term "pathogen" refers to an organism that adversely affects plants by colonizing, damaging, attacking, or infecting them. Pathogens can therefore affect plant growth, development, reproduction, harvest, or yield. This includes organisms that spread disease and / or damage the host and / or compete with the host for nutrients. Plant pathogens include, but are not limited to, fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoans, nematodes, insects, and parasitic plants.

[0704] Non-limiting examples of pathogens include roundheaded borers, such as the long horned borer; psyllids, such as the red gum lerp psyllid (Glycaspis brimblecombei), blue gum psyllid, spotted gum lerp psyllid, and lemon gum lerp psyllid; tortoise leaf beetles; weevils; leaf beetles; armillaria; Thaumastocoris peregrinus; and the eucalyptus wasp (Leptocybe invasa). These include, but are not limited to, sessile gall wasps (Cynipidae) such as Ophelimus maskelli and Selitrichodes globules; leaf-eating caterpillars such as the Omnivorous looper and the Orange tortrix; the Glassy-winged sharpshooter; and whiteflies such as the Giant whitefly. Other non-limiting examples of pathogens include aphids such as Chaitophorus species, Cloudywinged cottonwood and Periphyllus species; Armored scale insects such as the Apple persimmon scale and the San Jose scale. scale; carpenterworm; clearwing moth borers such as the American hornet moth and the Western poplar clearwing; flatheaded borers such as the bronze birch borer and the bronze poplar borer; folivorous caterpillars such as the fall webworm, fruit-tree leafroller, redhumped caterpillar, willow tussock moth, yellow nymph, tent caterpillar, tussock moth, and western tiger swallowtail; leafminers such as the poplar shield bearer; cottonwood gall mites Gall aphids such as the poplar petiolegall aphid; glassy-winged sharpshooters; leaf beetles and flea beetles; mealybugs; poplar and willow borers; longhorn beetles; sawflies; scale insects such as the olive scale, the Japanese holly scale, the maple boll scale, and the European fruit lecanium; treehoppers such as the buffalo treehopper; and hemipteran insects such as earwigs and lygus bugs. ...

Claims

1. 1. A method for producing an RNA molecule having silencing activity in a cell, comprising: (a) identifying nucleic acid sequences encoding RNA molecules that exhibit a predetermined range of sequence homology, not including complete identity, to nucleic acid sequences encoding RNA molecules that associate with an RNA-induced silencing complex (RISC); (b) determining the transcription of the nucleic acid sequences encoding the RNA molecules to select transcribable nucleic acid sequences encoding the RNA molecules that exhibit the predetermined sequence homology range; (c) determining the processivity into small RNAs of transcripts of the transcribable nucleic acid sequences encoding the RNA molecules exhibiting the predetermined sequence homology range to select the transcribable nucleic acid sequences encoding the RNA molecules exhibiting the predetermined sequence homology range, wherein the RNA molecules are aberrantly processed; (d) modifying the nucleic acid sequence of the transcribable nucleic acid sequence encoding the aberrantly processed RNA molecule exhibiting the predetermined sequence homology range to confer association with RISC and processibility to a small RNA complementary to a first target RNA; thereby generating an RNA molecule having silencing activity in the cell; A method comprising:

2. The method of claim 1, wherein the RNA molecule of step (a) encoded by the identified nucleic acid sequence exhibits a predetermined range of sequence homology, which does not include complete identity, to an RNA molecule associated with RISC and / or processed into a molecule associated with RISC.

3. The method of claim 1 or 2, wherein imparting processivity in step (d) comprises imparting standard processing to an RNA molecule encoded by a nucleic acid sequence of the nucleic acid sequence encoding an RNA molecule that associates with an RNA-induced silencing complex (RISC).

4. The method of any one of claims 1 to 3, further comprising determining the genomic location of the nucleic acid sequence encoding the RNA molecule that exhibits the predetermined sequence homology range of step (a).

5. 5. The method of claim 4, wherein the genomic location is within a non-coding gene, optionally within an intron of the non-coding gene.

6. 5. The method of claim 4, wherein the genomic location is within a coding gene, optionally within an exon of a coding gene, optionally within an exon encoding an untranslated region (UTR) of a coding gene, or optionally within an intron of a coding gene.

7. 7. The method of any one of claims 1 to 6, wherein steps (b) and / or (c) are affected by aligning small RNA expression data to the genome of the cell and determining the amount of reads that map to each genomic location.

8. 8. The method of claim 7, wherein the alignment of the small RNA is to a predetermined location in the genome of the cell without any mismatches.

9. 9. The method of any one of claims 1 to 8, wherein the modification of the nucleic acid sequence of the transcribable nucleic acid sequence confers structure to the aberrantly processed RNA molecule, such that the RNA molecule is processed into small RNAs that associate with RISC.

10. 10. The method of any one of claims 1 to 9, wherein the modification of the nucleic acid sequence of the transcribable nucleic acid sequence encoding the aberrantly processed RNA molecule exhibiting the predetermined sequence homology range is performed on a nucleic acid other than that corresponding to a binding site for the first target RNA.

11. 11. The method of any one of claims 1 to 10, wherein the processivity is mediated by a cellular nuclease selected from the group consisting of Dicer, Argonaute, tRNA cleavage enzymes, and Piwi-binding RNA (piRNA) associated proteins.

12. 12. The method of any one of claims 1 to 11, wherein the modification in step (d) comprises introducing into the cell a DNA editing agent that reactivates the silencing activity of the aberrantly processed RNA molecule toward the first target RNA, thereby generating an RNA molecule having silencing activity in the cell.

13. 13. The method of any one of claims 1 to 12, further comprising altering the specificity of the RNA molecule having silencing activity in the cell, wherein the DNA editing agent redirects the silencing specificity of the RNA molecule towards a target RNA of interest that is different from the first target RNA, thereby altering the specificity of the RNA molecule having the silencing activity in the cell.

14. 14. The method of any one of claims 1 to 13, wherein the nucleic acid sequence encoding the RNA molecule identified in step (a) is homologous to a gene encoding a silencing RNA molecule whose silencing activity and / or processing into small silencing RNAs depends on its secondary structure.

15. 15. The method of claim 14, wherein the silencing RNA molecule, whose silencing activity and / or processing into small silencing RNA depends on secondary structure, is selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA or U-RNA), small nucleolar RNA (snoRNA), small Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), RNA derived from repeats, RNA derived from autonomous and non-autonomous transposable and retrotransposable elements, RNA of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNA (lncRNA).

16. A genetically modified cell comprising a genome containing a polynucleotide sequence encoding an RNA molecule having a nucleic acid sequence change that causes the RNA molecule to be processed into a small RNA that associates with RISC, wherein said processing of the RNA molecule is not present in a wild-type cell of the same origin that does not have the nucleic acid sequence change.

17. 17. The genetically modified plant of claim 16, wherein the processing is standard processing.

18. 18. The genetically modified cell of claim 16 or 17, wherein the RNA molecule has silencing activity.

19. The RNA molecule may be a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi-binding RNA (piRNA), a phased small interfering RNA (phasiRNA), a trans-acting siRNA (tasiRNA), a transfer RNA fragment (tRF), a small nuclear RNA (snRNA), a translocating and / or relay RNA (tRF).

19. The method of any one of claims 1 to 13 or the genetically modified cell of any one of claims 16 to 18, wherein the RNA is selected from the group consisting of RNAs of retrotransposable origin, autonomous and non-autonomous transposable and / or retrotransposable RNAs.

20. 20. The method of any one of claims 1 to 15 or 19, further comprising introducing a donor oligonucleotide into the cell.

21. 21. The method of any one of claims 12-15, 19, or 20, wherein the DNA editing agent comprises at least one sgRNA.

22. 22. The method of any one of claims 12-15, 19-20, or 21, wherein the DNA editing agent does not comprise an endonuclease.

23. 22. The method of any one of claims 12-15, 19-20, or 21, wherein the DNA editing agent comprises an endonuclease.

24. 24. The method of any one of claims 12 to 15 or 19 to 23, wherein the DNA editing agent is a DNA editing system selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR endonucleases, dCRISPR endonucleases, and homing endonucleases.

25. 25. The method of any one of claims 23 or 24, wherein the endonuclease comprises Cas9.

26. 26. The method of any one of claims 12-15 or 19-25, wherein the DNA editing agent is applied to the cell as DNA, RNA, or RNP.

27. 27. The method of any one of claims 13 to 15 or 19 to 26, wherein the target RNA of interest is endogenous or exogenous to the cell.

28. 28. The method of any one of claims 13 to 15 or 19 to 27, wherein the specificity of the RNA molecule is determined phenotypically by determining at least one phenotype selected from the group consisting of cell size, rate / inhibition of growth, cell shape, cell membrane integrity, tumor size, tumor shape, organism coloration, organism size, crop yield, metabolic profile, fruit traits, biotic stress resistance, abiotic stress resistance, infection parameters, and inflammation parameters.

29. 29. The method of any one of claims 13 to 15 or 19 to 28 or the genetically modified cell of any one of claims 16 to 18 or 19, wherein the cell is a eukaryotic cell.

30. 30. The method or genetically modified cell of claim 29, wherein the eukaryotic cell is obtained from a eukaryotic organism selected from the group consisting of plants, mammals, invertebrates, insects, nematodes, birds, reptiles, fish, crustaceans, fungi, and algae.

31. 30. The method or genetically modified cell of claim 29, wherein the eukaryotic cell is a plant cell.

32. 32. The method of claim 31 , wherein the plant cell is a protoplast. Cells.

33. 33. A plant cell produced according to the method of any one of claims 1 to 15 or 19 to 32.

34. 34. A plant comprising the plant cell of claim 33.

35. 35. The plant of claim 34, wherein the plant is non-transgenic.

36. 1. A method for producing a plant with reduced expression of a target gene, comprising: (a) breeding a plant according to claim 34 or 35; (b) selecting progeny plants that have reduced expression of the target RNA of interest or that contain a silencing agent in the RNA molecule specific for the target RNA of interest and that do not contain the DNA editing agent; thereby producing the plant having reduced expression of the target gene; A method comprising:

37. 1. A method for generating a plant comprising an RNA molecule having silencing activity against a target RNA of interest, comprising: (a) breeding a plant according to claim 34 or 35; (b) selecting progeny plants comprising the RNA molecule having the silencing activity for the target RNA of interest, or comprising a silencing activity in the RNA molecule specific for the target RNA of interest, and not comprising the DNA editing agent; thereby generating a plant comprising an RNA molecule having silencing activity against a target RNA of interest; A method comprising:

38. 36. A method of producing a plant or plant cell according to claim 34 or 35, comprising growing the plant or plant cell under conditions which allow reproduction.

39. 38. The method of claim 36 or 37, wherein the breeding comprises crossing or selfing.

40. 40. Seeds of a plant according to any one of claims 34 or 35 or a plant produced according to any one of claims 36 to 39.

41. 30. The method or genetically modified cell of claim 29, wherein the eukaryotic cell is a human cell.

42. 42. The method or genetically modified cell of claim 41, wherein the nucleic acid sequence encoding the RNA molecule is selected from the group consisting of the nucleic acid sequences set forth in any of SEQ ID NOs: 352-392.

43. 43. The method or genetically modified cell of claim 41 or 42, wherein the eukaryotic cell is a totipotent stem cell.

44. 10. A method of treating a disease in a subject in need thereof, comprising generating an RNA molecule with silencing activity and / or specificity according to the method of any one of claims 1 to 15, 19 to 32 or 41 to 43, wherein said RNA molecule has silencing activity for a transcript of a gene associated with the onset or progression of said disease, thereby treating said subject.

45. 1. A method of introducing silencing activity into a first RNA molecule in a cell, comprising: (a) i. a first nucleic acid sequence is transcribed into said first RNA molecule within the cell; ii. the sequence of the first RNA molecule has partial homology, except for sequence identity, to the sequence of a second RNA molecule, the second RNA molecule being processible into a third RNA molecule having silencing activity, and the second RNA molecule being encoded by a second nucleic acid sequence in the cell; and iii. The first RNA molecule is non-processible or processible differently from the second RNA molecule, such that the first RNA molecule is not processed into an RNA molecule having the same silencing activity as the third RNA molecule; selecting the first nucleic acid sequence in the cell; (b) modifying the first nucleic acid sequence to encode a modified first RNA molecule, wherein the modified first RNA molecule is processible into a fourth RNA molecule in the same manner as the second RNA molecule is processible into the third RNA molecule, such that the fourth RNA molecule has the same qualitative silencing activity as the third RNA molecule; thereby introducing silencing activity into said first RNA molecule; A method comprising:

46. 46. ​​The method of claim 45, wherein the second RNA molecule is an RNA molecule having a secondary structure that allows it to be processed into an RNA having silencing activity, and optionally, the silencing activity is mediated through association with RISC.

47. 47. The method of claim 46, wherein the RNA molecule having a secondary structure that enables it to be processed into an RNA with silencing activity is selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA or URNA), small nucleolar RNA (snoRNA), small Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), RNA derived from repeats, RNA derived from autonomous and non-autonomous transposable and retrotransposable elements, RNA of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNA (lncRNA).

48. 47. The method of claim 46, wherein the first nucleic acid sequence results in a secondary structure that allows the modified first RNA molecule to be processed into a fourth RNA molecule.

49. 49. The method of claim 48, wherein modifying the first nucleic acid sequence comprises modifying the sequence such that the modified first RNA molecule has essentially the same secondary structure as the second RNA molecule, optionally a secondary structure that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical to the secondary structure of the second RNA molecule.

50. The method of claim 45, wherein the first nucleic acid molecule is a gene derived from human (H. sapiens) and selected from the group consisting of genes having a sequence set forth in any one of SEQ ID NOs: 352 to 392.

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