Method and composition for generating dominant alleles using genome editing

Abstract

The present disclosure provides methods and compositions for generating dominant alleles using targeted editing techniques. Modified chromosomes, cells, tissues, and plants containing the modified dominant alleles are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 854,142, filed May 29, 2019; U.S. Provisional Application No. 62 / 886,726, filed Aug. 14, 2019; and U.S. Provisional Application No. 62 / 886,732, filed Aug. 14, 2019, which are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to methods and compositions for generating dominant alleles via targeted editing of genomes.

[0003] Incorporation of Sequence Listing The sequence listing contained in the file named "P34497WO00_SL.TXT", created on May 28, 2020, 172,842 bytes (measured in MS - Windows®), is electronically submitted with this specification and is hereby incorporated by reference in its entirety.

Background Art

[0004] A dominant allele is an allele that suppresses the contribution of a second allele at the same locus. A dominant allele can be either a dominant - negative allele or a dominant - positive allele. A dominant - negative allele, or antimorph, is an allele that acts in opposition to normal allele function. For example, a dominant - negative allele often interferes with the normal function of an allele in a heterozygous or homozygous state. A dominant - positive allele can increase normal gene function (e.g., hypermorph) and / or confer a broader or new function to the gene (e.g., neomorph).

[0005] Natural and random mutagenesis techniques (e.g., ethylmethyl sulfonate and T-DNA insertion) have been used to generate mutations in various cell types. However, dominant mutations occur infrequently and are difficult to obtain in a given gene of interest. Therefore, methods and compositions for selectively editing genomes to produce dominant-negative or dominant-positive alleles would be beneficial. [Overview of the project]

[0006] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising using targeted editing techniques to invert a portion of the gene and generate an antisense RNA transcript that can induce repression of the unmodified allele.

[0007] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising deleting a portion of a chromosome between a first gene region and a second gene region using a targeted editing technique, wherein an antisense RNA transcript of the first gene region is generated after the deletion of the portion of the chromosome.

[0008] In one embodiment, the Disclosure provides a method for generating a dominant-negative allele of a gene in one or more cells, comprising: (a) inducing a first double-strand break and a second double-strand break that flank in the targeting region of the gene; (b) identifying one or more cells containing an inversion of the targeting region of the gene, wherein the inversion results in the production of an antisense RNA transcript from the targeting region; and (c) selecting one or more cells containing the inversion of the targeting region of the gene.

[0009] In one embodiment, the Disclosure provides a method for reducing protein expression in cells, comprising (a) inducing a first double-strand break and a second double-strand break that flank a chromosomal targeting region, and (b) identifying one or more cells containing an inversion in the chromosomal targeting region, wherein protein expression is reduced compared to control cells that do not contain an inversion in the targeting region.

[0010] In one embodiment, the Disclosure provides a method comprising (a) identifying a chromosomal region comprising a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region and the first and second promoters are positioned in opposite directions; (b) inducing a first double-strand break and a second double-strand break that flank a targeting region; (c) identifying one or more cells comprising a deletion of a chromosomal targeting region; and (d) selecting one or more cells comprising a deletion of a chromosomal targeting region.

[0011] In one embodiment, the Disclosure provides a method for reducing gene expression in at least one cell, comprising: (a) inducing a double-strand break at a target site of a gene using a targeted editing technique; (b) inserting a donor sequence at the double-strand break, wherein the donor sequence includes a tissue-specific or tissue-preferential promoter and is inserted at the target site such that the tissue-specific or tissue-preferential promoter is reversed relative to the gene; and (b) identifying at least one cell containing the reversed donor sequence insertion, wherein gene expression is reduced compared to a control cell without the donor sequence insertion.

[0012] In one embodiment, the Disclosure provides a method for modifying gene expression, comprising: (a) inducing a double-strand break at a target site using a targeted editing technique; (b) inserting a donor sequence at the double-strand break, wherein the donor sequence comprises an endogenous element (e.g., a promoter, enhancer, or promoter / enhancer fragment) or a designed element capable of inducing increased or ectopic expression of the gene; and (c) identifying at least one cell containing the donor sequence, wherein the expression of the target gene is increased in at least one tissue compared to control cells not containing the donor sequence.

[0013] In one embodiment, the Disclosure provides a method for promoting gene expression, comprising: (a) inducing a double-strand break at a target site using a targeted editing technique; (b) inserting a donor sequence at the double-strand break, wherein the donor sequence comprises an endogenous element (e.g., a promoter, enhancer, or promoter / enhancer fragment) or a designed element capable of inducing increased or ectopic expression of the gene; and (c) identifying at least one cell containing the donor sequence, wherein the expression of the target gene is increased in at least one tissue compared to control cells not containing the donor sequence.

[0014] In one embodiment, the Disclosure provides a method for generating a dominant-positive allele, comprising: (a) inducing a double-strand break at a target site using a targeted editing technique; (b) inserting a donor sequence at the double-strand break, wherein the donor sequence comprises a sequence of an endogenous gene; and (c) identifying at least one cell containing the donor sequence, wherein gene expression is increased in at least one tissue compared to control cells not containing the donor sequence.

[0015] In one embodiment, the present disclosure provides a method for reducing gene expression in a cell, comprising: (a) identifying a chromosomal region comprising a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region, and the first and second promoters are positioned in opposite directions; (b) inducing a first double-strand break and a second double-strand break that flank a target region using a targeted editing technique, wherein the target region comprises a second coding region and an intervening region; and (c) identifying one or more cells containing a deletion of the target region, wherein the second promoter generates at least one antisense RNA of the first coding region, and the expression of the first coding region is reduced compared to control cells that do not contain the deletion of the target region.

[0016] In one embodiment, the present disclosure provides a method for reducing the expression of a protein of interest in cells, comprising: (a) identifying a chromosomal region containing a gene region encoding the protein of interest, including a first promoter and a protein coding region, and a second chromosomal region containing a second promoter and an intervening region, wherein the protein coding region and the second promoter of interest are separated by the intervening region and the first and second promoters are positioned in opposite directions; (b) inducing a first double-strand break and a second double-strand break that flank the intervening region using a targeted editing technique; and (c) identifying one or more cells containing a deletion of the intervening region, wherein the expression of the protein of interest is reduced compared to control cells that do not contain the deletion of the intervening region.

[0017] In one embodiment, the Disclosure provides a method for generating inversions in a targeting region of a gene, comprising: (a) providing one or more cells with at least one RNA guide nuclease, or one or more vectors encoding at least one RNA guide nuclease, wherein the at least one RNA guide nuclease flanks at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, and at least The present invention provides a method comprising: (b) identifying one or more cells containing an inversion in the targeting region of a gene, wherein the inversion results in the production of an antisense RNA transcript from the targeting region; and (c) selecting one or more cells containing an inversion in the targeting region of a gene.

[0018] (a) Identifying a chromosomal region comprising a first gene region including a first promoter and a first coding region, and a second gene region including a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region, and the first and second promoters are positioned in opposite directions; and (b) Providing one or more cells with at least one RNA guide nuclease, or one or more vectors encoding at least one RNA guide nuclease, wherein at least one RNA guide nuclease is a chromosomal mark A method comprising (c) identifying one or more cells containing a deletion of a targeting region, and (d) selecting one or more cells containing a deletion of a targeting region.

[0019] In one embodiment, the Disclosure provides a method for (a) providing one or more cells to one or more cells comprising at least one RNA guide nuclease and at least one donor molecule, or one or more vectors encoding at least one RNA guide nuclease and at least one donor molecule, wherein at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site of at least one gene, the donor molecule comprises a designed element, the RNA guide nuclease produces a double-strand break at the target site, and the designed element is inserted by the double-strand break; and (b) identifying one or more cells containing the insertion of the designed element at the target site; and (c) selecting one or more cells containing the insertion of the designed element at the target site.

[0020] In one embodiment, the disclosure provides (a) one or more cells with a vector encoding at least one RNA guide nuclease and at least one donor molecule, or at least one RNA guide nuclease and at least one donor molecule, wherein the at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site of at least one gene, and the donor molecule is tissue-specific. The present invention provides a method comprising: (b) identifying one or more cells containing an insertion at the target site of a sequence encoding a tissue-specific or tissue-preferential promoter such that the sequence encoding the tissue-specific or tissue-preferential promoter is oriented in the opposite direction to the gene; and (c) selecting one or more cells containing an insertion at the target site of a sequence encoding a tissue-specific or tissue-preferential promoter.

[0021] In one embodiment, the Disclosure provides a method comprising: (a) providing one or more RNA guide nucleases, or one or more vectors encoding one or more RNA nucleases, to one or more cells, wherein one or more RNA guide nucleases can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site, and the one or more RNA guide nucleases produce a double-strand break at the target site; (b) identifying at least one cell containing an insertion or deletion at a target site, wherein the insertion or deletion at the target site results in the generation of a dominant-negative allele of at least one gene; and (c) selecting one or more cells containing a dominant-negative allele of at least one gene.

[0022] In one embodiment, the Disclosure provides a method comprising: (a) providing one or more RNA guide nucleases, or one or more vectors encoding one or more RNA nucleases, to one or more cells, wherein one or more RNA guide nucleases can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site, and the one or more RNA guide nucleases produce a double-strand break at the target site; (b) identifying at least one cell containing an insertion or deletion at a target site, wherein the insertion or deletion at the target site results in the generation of a dominant-positive allele of at least one gene; and (c) selecting one or more cells containing a dominant-positive allele of at least one gene.

[0023] In one embodiment, the Disclosure provides a method comprising (a) using targeted editing techniques to generate a first double-strand break (DSB) and a second DSB in a first allele of a gene in a cell; (b) using targeted editing techniques to generate a third DSB in a second allele of a gene in a cell; and (c) identifying a cell containing an insertion of a region of the first allele in the reverse direction at the site of the third DSB in the second allele, thereby generating a modified second allele.

[0024] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of a gene, comprising using a targeted editing technique to introduce at least one non-coding RNA target site into the gene.

[0025] In one embodiment, the disclosure provides a method for generating a dominant allele of a gene, comprising using targeted editing techniques to introduce a nonsense mutation into a gene to produce a cleaved protein or a protein in which the amino acid composition downstream of the mutation is altered. This technique can also be combined with a second targeted editing mutation to restore the frame of the normal amino acid sequence downstream of the initial mutation and create a nonsense region within the polypeptide.

[0026] In one embodiment, the Disclosure provides a method comprising: (a) providing cells with an engineered pentatricopeptide repeat (PPR) protein, or a vector encoding an engineered PPR protein operably linked to a promoter, such that the engineered PPR protein can bind to an RNA transcript of a target gene; (b) selecting one or more cells expressing the engineered PPR protein from step (a); and (c) identifying one or more cells selected in step (b) that exhibit altered expression of the target gene.

[0027] In one aspect, the present disclosure provides a method for generating a dominant negative allele of a gene in a cell, which includes using a targeted editing technique to insert an inverted copy of a gene or a portion thereof adjacent to the native copy of the gene to generate an inverted repeat sequence capable of producing an antisense RNA transcript of the gene or a portion thereof.

[0028] In one aspect, the present disclosure provides a method for generating a dominant negative or dominant positive allele of a gene in a cell, which includes using a targeted editing technique to delete a portion of the gene, wherein a microprotein is generated after the deletion of the portion of the gene.

[0029] In one aspect, the present disclosure provides a method for generating a dominant negative or dominant positive allele of a gene in a cell, which includes using a targeted editing technique to delete a portion of the intergenic region, wherein the gene is placed under the control of an upstream promoter by the deletion. In some embodiments, the upstream promoter drives an increase in the expression of the gene. In some embodiments, the upstream promoter drives a decrease in the expression of the gene. In some embodiments, the upstream promoter drives a change in the temporal expression of the gene. In some embodiments, the upstream promoter drives a change in the tissue-specific expression of the gene.

[0030] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of at least one gene in at least one cell, comprising: (a) introducing into at least one cell a genome editing system comprising (i) a site-specific nuclease or a molecule encoding a site-specific nuclease, (ii) a single guide RNA (sgRNA) or a molecule encoding an sgRNA, and (iii) one or more molecules encoding at least a first tether-guide oligo (tgOligo) and a second tgOligo, or the first and second tgOligo, operably ligated to at least one promoter; and (b) generating a first double-strand break (DSB) and a second DSB in at least one gene. The present invention provides a method comprising (c) identifying and selecting at least one cell containing the cleaved protein, wherein a first tgOligo and a second tgOligo hybridize to the 3' free ends of opposing strands in the first DSB and the second DSB, resulting in the deletion of at least one, at least two, at least three, at least four, at least five, at least ten, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 2500, or at least 5000 nucleotides of at least one gene, thereby generating a dominant-negative allele of a gene encoding a cleaved protein.

[0031] In one embodiment, the present disclosure is a method for generating a dominant-negative allele of at least one gene in at least one cell, comprising: (a) introducing one or more vectors into at least one cell that encode (i) at least one site-specific nuclease, (ii) at least one single guide RNA (sgRNA), and (iii) at least one promoter operably linked to at least one tether-guide oligo (tgOligo) and a second tgOligo; and (b) generating a first double-strand break (DS) in the gene. The present invention provides a method comprising (b) generating a second DSB, wherein the first tgOligo and the second tgOligo hybridize to the 3' free ends of opposing strands in the first and second DSBs, so that the region of at least one gene between the first and second DSBs is oriented in opposite directions, thereby generating a dominant-negative allele of at least one gene encoding an antisense RNA transcript of the gene, and (c) identifying and selecting at least one cell containing the antisense RNA transcript of at least one gene.

[0032] In one embodiment, the disclosure provides a modified plant cell comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript comprising a sequence complementary to the native transcript sequence of the gene or a portion thereof.

[0033] In one embodiment, the disclosure provides a modified chromosome comprising a non-transposon-mediated deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript comprising a sequence complementary to the native transcript sequence of the gene or a portion thereof.

[0034] In one embodiment, the disclosure provides a modified plant or part thereof comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript comprising a sequence complementary to the native transcript sequence of the gene or a portion thereof.

[0035] In one embodiment, the disclosure provides a modified cell comprising (a) a non-transposon-mediated genomic deletion of at least one gene or a portion thereof at an endogenous locus of at least one gene, or (b) a non-transposon-mediated and non-T-DNA-mediated insertion of a polynucleotide sequence into at least one gene, wherein the deletion or insertion produces a dominant-positive allele of at least one gene.

[0036] In one embodiment, the disclosure provides a modified cell comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of at least one gene, wherein the deletion or inversion results in the production of an RNA transcript comprising a sequence complementary to the native transcript sequence of the gene.

[0037] In one embodiment, the disclosure provides a modified cell comprising targeted editing of at least one gene or a portion thereof, wherein the targeted editing generates an RNA transcript complementary to the native transcript sequence of the gene.

[0038] In one embodiment, the disclosure provides a modified cell comprising at least one dominant-negative allele of at least one gene generated by a targeted editing technique, wherein, upon transcription of the at least one dominant-negative allele, the allele generates an RNA transcript capable of forming a hairpin loop secondary structure.

[0039] In one embodiment, the disclosure provides a modified cell comprising a non-transgenic dominant-negative allele of a gene, wherein the dominant-negative allele comprises a heterologous non-coding RNA target site at an endogenous locus of the gene.

[0040] In one embodiment, the disclosure provides a modified cell comprising a non-transgenic dominant-positive allele of a gene, wherein the dominant-positive allele comprises a heterologous non-coding RNA target site at an endogenous locus of the gene.

[0041] In one embodiment, the disclosure provides a modified cell comprising at least one insertion or deletion at an endogenous locus of at least one gene generated by a targeted editing technique, wherein the insertion or deletion results in the expression of a cleavage protein.

[0042] In one embodiment, the disclosure provides a modified cell comprising a dominant-negative allele of at least one gene and an inverted copy of the gene adjacent to the native copy of the gene at the endogenous locus of the gene. [Brief explanation of the drawing]

[0043] [Figure 1] This includes panels A and B. Panel A shows that two sRNAs coupled to two RNA guide nucleases can hybridize target DNA on the same or different strands, generating two double-strand breaks (DSBs) and separating a region of DNA from the rest of the DNA strand. Panel B shows various possible outcomes after two RNA guide nucleases have generated two DSBs in DNA. Native cellular mechanisms can repair the two DSBs by deleting the entire region between the DSBs, deleting only a small portion of the region from the 5' or 3' end, or inverting the region between the DSBs. [Figure 2]The panel includes panels A and B. Panel A shows the representation of a maize genome region near the GA20 oxidase_5 gene. As shown in panel A, the methyltransferase / SAMT gene is adjacent to the GA20 oxidase_5 gene but oriented in the opposite direction. The intervening region between the SAMT promoter and the GA20 oxidase_5 coding gene is deleted after generating double-strand breaks at each end. Panel B shows the structure of the region after deletion of the intervening region. By removing the intervening region, the SAMT promoter can drive the expression of an antisense GA20 oxidase_5 RNA transcript, which can form a double-strand RNA with the sense GA20 oxidase_5 RNA transcript produced by the native GA20 oxidase_5 promoter. [Figure 3] The panel includes panels A, B, C, and D. Panel A shows GUS staining of Arabidopsis thaliana plants, indicating that the native 3 promoter expresses GUS only in root tissue. Panel B shows the genomic structure around the native 3 promoter and GUS transgene. Panel C shows expanded GUS staining after insertion of a native genomic expression element, such as an enhancer element or a designed expression element, upstream of the native 3 TATA box. Panel D shows the genomic structure around the native 3 promoter after targeted insertion of a native enhancer or designed element. [Figure 4] The panel includes panels A, B, C, and D. Panel A shows the structure of the gene of interest and demonstrates that a double-strand break can be generated immediately upstream of the polyadenylation site in the 3'-UTR of the gene of interest. Panel B shows the insertion of an antisense promoter at the double-strand break site. The promoter can be the gene's native promoter or any promoter of interest. Panel C shows the optional generation of two double-strand breaks surrounding the native promoter of the gene of interest, which results in its deletion (Panel D). [Figure 5]The panel includes panels A, B, C, and D. Panel A shows GUS staining in Arabidopsis thaliana plants containing a GUS transgene under the control of the native 3 promoter. GUS is expressed throughout the plant. Panel B shows the genomic region around the native 3 promoter and the GUS transgene. Panel C shows the reduction in GUS staining when a leaf-specific promoter is inserted downstream of the GUS transgene to transcribe antisense GUS RNA. Panel D shows the genomic region around the native 3 promoter and the GUS transgene after insertion of the antisense leaf-specific promoter. [Figure 6] The following diagrams illustrate the possible consequences of protein cleavage. The top flow shows typical protein interactions, where all encoded components are brought together in the correct three-dimensional structure, enabling the protein complex to function. The middle flow shows the cleavage of one protein component, which removes the functional unit of the protein but retains the interaction domain, which can still bind to the interacting protein and block the interaction site from the fully functional cleaved protein. In the middle example, this acts as a dominant-negative allele. The bottom flow shows strategic deletion of a portion of a protein, which encodes an activity-regulating element and results in a protein complex that remains in either a constitutively active or repressed state of activity by leaving the functional and interaction domains intact. [Figure 7] The panel includes panels A, B, C, D, and E. Panel A shows heterozygous genomic loci. Panel B shows that a nuclease (represented by scissors) produces one DSB on the first allele. Panel C shows that a nuclease produces two DSBs on the second allele. In one possible outcome (panel D), the region between the two DSBs on the second allele (panel C) is inverted and inserted into a single DSB on the first allele. Panel E shows the hairpin RNA transcript production morphology transcribing the allele structure of panel D. [Figure 8]A schematic diagram of the insertion of a non-coding RNA target site in the target gene is provided. Such insertions can lead to the production of transient secondary siRNAs that downregulate the target gene (GOI). [Figure 9] A schematic diagram of a Cas9-mediated double-strand break (DSB) and a tether guide oligo (tgOligo) bound to a target DNA site is provided. The Cas9-PAM interaction occurs on the non-target strand, and sgRNA-DNA annealing occurs on the target strand. The blunt end of the Cas9 break site is held in place by Cas9 at the 5' end of the non-target strand (PAM site) and both break ends (3' and 5') of the target strand. The 3' break end of the non-target strand is free and "flaps". The 3' free "flap" end of the non-target strand can be up to 35 nucleotides, which may be sufficient for specific complementar binding. A tgOligo (e.g., an ssDNA template) can be included for the incorporation of desired nucleotide modifications. The drawing scheme used here follows in subsequent figures. [Figure 10] Cas9 is shown conjugated to a homodimer domain (top) and a heterodimer domain (center and bottom right) to facilitate dimerization. The ligands for the homodimer and heterodimer domains are shown (bottom left). The drawing schemes used here for ligands, homodimer or heterodimer domains, ssDNA binding domains, etc., are shown in subsequent figures. The components of the Cas9 / sgRNA complex and target DNA are shown as illustrated in Figure 9. The drawing schemes for the various dimerization domains used here are shown in subsequent figures. [Figure 11] This paper demonstrates the use of catalytically inactivated Cas9 (dCas9) to increase genome editing efficiency. Panel 1 illustrates how dCas9 binds to DNA at a target site designated by gRNA, creating a loop structure accessible by template-based editing. Panel 2 illustrates a modified scheme to further facilitate template-based editing via dCas9 conjugated with an ssDNA binding domain. The editing efficiency of this modified scheme is expected to be higher than that of Panel 1, as the ssDNA template binds to the dCas9 complex and is closer to the gRNA target. [Figure 12] This document provides an example of a construct containing Cas9, gRNA, and tgOligo. RZ stands for ribozyme, an enzyme that cleaves the 15bp recognition site (RZ site) within RNA. [Figure 13] This document provides examples of various approaches to improve genome editing efficiency. Using dimerization domains (see Figure 10), tgOligo (see Figure 9), or a combination of both, recovery of complete knockout (deletion) of genomic regions flanked by two gRNA target sites can be facilitated. Panel 1 shows knockout (KO) events facilitated by dimerization. Panel 2 shows KO events facilitated by tgOligo. Panel 3 shows KO events facilitated by a combination of dimerization and tgOligo. Panel 4 shows inversion events facilitated by tgOligo. Panel 5 shows inversion events facilitated by dimerization. Panel 6 shows inversion events assisted by a combination of Cas9 dimerization / inactivation and tgOligo. Only configurations where two gRNAs recognize different strands of the target dsDNA are shown. The same concept is equally applicable to other configurations where two gRNAs recognize the same strand of the target dsDNA. [Figure 14]This paper provides an example of dominant knockout allele generation via genomic inversion through editing of the maize BR2 gene. Two exemplary gRNAs are used. The first gRNA (shown on the left) targets the end of the first exon of BR2, and the second gRNA (shown on the right) recognizes the start codon region of the adjacent GRMZM2G491632 gene. Inversion of the genomic segment flanked by these two gRNAs can result in a BR2 antisense partial transcript (see Transcript 1). This BR2 antisense transcript is produced via GRMZM2G491632 promoter activity. By modulating the relative positions of the two gRNAs, a BR2 antisense full transcript (e.g., by moving the first gRNA on the left to target the start codon region of the BR2 gene) or a BR2 antisense transcript under the control of the native BR2 promoter (e.g., by moving the second gRNA on the right to target the stop codon region of the BR2 gene) can be achieved. [Figure 15] Examples are provided of template-based editing or site-directed integration (SDI) facilitated by dimerization at a single location (Panels 1 and 2) or multiple locations (Panel 3), and template-based editing or SDI facilitated by dimerization / tgOligo (Panel 4). [Figure 16] Examples of template editing (Panel 1), site-directed embedding (Panel 2), and / or recombination (Panel 3) using tgOligo are provided. [Figure 17] This provides an example of head-to-tail stacking of inverted Y1 genes to produce antisense transcripts and silence gene expression. This approach can create a dominant mutant Y1 allele for normally recessive traits, which remains under the control of the native Y1 promoter. [Figure 18]Examples of microproteins are presented. Microproteins often target transcription factors that bind to DNA as active homodimers. Microproteins interfere with their targets by forming non-functional heterodimer complexes that cannot bind to DNA. The DBD is the DNA-binding domain, and the PPI is the protein-protein interaction domain. [Figure 19] This includes panels A and B. Panel A shows the representation of a maize genome region near the MIR1 gene. As shown in panel A, the GRMZM2G150302 gene is located adjacent to and upstream of the GA20 oxidase_5 gene. The intervening region between the GRMZM2G150302 promoter and the MIR1 coding gene is deleted after generating double-strand breaks at each end. Panel B shows the structure of the region after deletion of the intervening region. By removing the intervening region, the GRMZM2G150302 promoter can drive the expression of the MIR1 gene. [Figure 20] This paper presents a concrete example of creating antisense RNA molecules targeting the Zm.GA20ox5 and Zm.GA20ox3 genes by deleting the genomic region between Zm.GA20ox5 and its neighboring gene Zm.SAMT, which is oriented in the opposite direction, via genome editing. [Figure 21] This paper illustrates the genomic locations of various guide RNA target sites in three exemplary vectors for creating genomic deletions between the Zm.GA20ox5 gene and its neighbor, the Zm.SAMT gene. [Figure 22] The average height of wild-type plants and homozygous edited plants is shown in inches (Y-axis). [Figure 23] The average height of wild-type plants and homozygous or heterozygous edited plants is shown in inches (Y-axis). [Figure 24] The concentrations of GA12 and GA9 in the edited plants and control plants are shown in pmol / g (Y axis). [Figure 25] The concentrations of GA20 and GA53 in the edited plants and control plants are shown in pmol / g (Y axis). [Figure 26]The concentrations of active gibberellates GA1, GA3, and GA4 in the edited plants and control plants are shown in pmol / g (Y axis). [Figure 27] This paper presents specific examples of genomic modifications to the Zm.GA20ox3 locus that encode RNA transcripts having inverted sequences capable of hybridizing to the corresponding sequences of RNA transcripts to produce stem-loop structures, thereby causing repression of one or both copies or alleles of the endogenous Zm.GA20ox3 and Zm.GA20ox5 loci. [Figure 28] This shows the average height of wild-type and heterozygous edited maize plants. [Figure 29] This shows the number of 21-mer small RNAs per million reads (Y-axis) mapped to regions in the stem of edited stem-loops containing the inverted GA20ox5 sequence and the corresponding sequence of the edited GA20ox3 gene, detected in plant samples containing the edited GA20ox3 allele. [Figure 30] The concentrations of GA12 and GA9 in edited maize plants and control maize plants are shown in pmole / g (Y axis). [Figure 31] The concentrations of GA20 and GA53 in edited maize plants and control maize plants are shown in pmole / g (Y axis). [Figure 32] The concentrations of GA1, GA3, and GA4 in edited maize plants and control maize plants are shown in pmole / g (Y axis). [Modes for carrying out the invention]

[0044] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Many methods may be used in the practice of this disclosure, as will be recognized by those skilled in the art. In fact, this disclosure is by no means limited to the methods and materials described. In this disclosure, the following terms are defined as follows:

[0045] This specification provides methods and compositions for generating dominant alleles using targeted editing techniques in a wide range of organisms, including plants, animals, fungi, and protists. A dominant allele is an allele that suppresses the contribution of a second allele at the same locus. A dominant allele may be a "dominant-negative allele" or a "dominant-positive allele." A dominant-negative allele, or antimorph, is an allele that acts in opposition to normal allele function. Dominant-negative alleles typically do not function normally and either directly inhibit the activity of the wild-type protein (e.g., via dimerization) or inhibit the activity of a second protein (e.g., an activator or downstream component of a pathway) required for the normal function of the wild-type protein. For example, a dominant-negative allele may interfere with or reduce the normal function of the allele in a heterozygous or homozygous state. A dominant-positive allele may increase or expand normal gene function (e.g., hypermorph) or confer new function to the gene (e.g., neomorph). Semi-dominant alleles arise when the penetrance of the associated phenotype in individuals heterozygous for the allele is lower than the penetrance observed in individuals homozygous for the allele.

[0046] Unless otherwise noted, the practices described herein utilize prior art in biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and biotechnology, which are within the scope of the art of those skilled in the art. Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Current Protocols In Molecular Biology (FMAusubel, et al. eds., (1987)), Series Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJMacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RIFreshney, ed. (1987)), Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences, CNStewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant Transformation See Technologies (Wiley-Blackwell) and RHSmith (2013) Plant Tissue Culture. Techniques And Experiments (Academic Press, Inc.).

[0047] All references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

[0048] As used herein, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise. For example, the terms "compound" or "at least one compound" may include multiple compounds, including mixtures of compounds.

[0049] When the term "and / or" is used in an enumeration of two or more items, it means that any one of the enumerated items may be used alone or in combination with any one or more of the enumerated items. For example, the expression "A and / or B" is intended to mean either A or B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0050] The nucleic acid molecules provided herein include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as their functional analogues such as complementary DNA (cDNA). The nucleic acid molecules provided herein may be single-stranded or double-stranded. Nucleic acid molecules contain the nucleotide bases adenine (A), guanine (G), thymine (T), and cytosine (C). In RNA molecules, uracil (U) replaces thymine. The symbol "N" can be used to represent any nucleotide base (e.g., A, G, C, T, or U). As used herein, "encode" means that a polynucleotide codes for an amino acid in a polypeptide. A sequence of three nucleotide bases codes for one amino acid. As used herein, "expressed," "express," or "express" means the transcription of RNA from a DNA molecule. As used herein, the terms "polypeptide," "peptide," and "protein" are used synonymously and refer to polymers of amino acid residues. This term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of the corresponding native amino acids. Messenger RNA, or mRNA, is an RNA transcript derived from polynucleotides that can be translated into proteins. Typically, DNA codes for mRNA, and mRNA codes for proteins. When DNA is transcribed by RNA polymerase to ultimately produce proteins, RNA polymerase typically produces a sense mRNA strand from an antisense DNA strand. The sense strand of DNA or RNA extends from 5' to 3', while the antisense strand extends from 3' to 5'. The sense and antisense strands of the same polynucleotide are complementary to each other.

[0051] In one embodiment, the nucleic acid molecules provided herein include nucleic acid molecules encoding proteins that are codon-optimized for eukaryotic cells. In another embodiment, the protein-encoding nucleic acid molecules are codon-optimized for plant cells. In yet another embodiment, the protein-encoding nucleic acid molecules are codon-optimized for monocotyledonous plant species. In a further embodiment, the protein-encoding nucleic acid molecules are codon-optimized for maize or soybean cells.

[0052] As used herein with respect to two or more nucleotide or protein sequences, the term “percent identity” or “percent identical” is calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) across a comparison window; (ii) determining the number of positions in which identical nucleic acid bases (in the case of nucleotide sequences) or amino acid residues (in the case of proteins) occur in both sequences to obtain the number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and (iv) multiplying this quotient by 100% to obtain the percentage identity. When “percent identity” is calculated in relation to a reference sequence without specifying a particular comparison window, the percentage identity is determined by dividing the number of matching positions in the alignment region by the total length of the reference sequence. Thus, in this application, when two sequences (query and subject) are optimally aligned (allowing gaps in their alignment), the “percent identity” of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the total length (or comparison window) of the query sequence and multiplying by 100%. When sequence identity percentages are used for proteins, it is recognized that non-identical residue positions are often due to conservative amino acid substitutions. Conservative amino acid substitutions are those in which an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. If multiple sequences differ due to conservative substitutions, the sequence identity percentage may be adjusted upward to compensate for the conservative nature of the substitutions. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity."

[0053] Various pairwise or composite sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool® (BLAST), are known in the art for performing optimal sequence alignment and calculating their percentage identity. These can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences. While other alignment and comparison methods are known in the art, the alignment and percentage identity (including the percentage identity range described above) between two sequences can be determined by the ClustalW algorithm. For example, Chenna R. et al., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31:3497-3500 (2003), Thompson JD et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22:4673-4680 (1994), Larkin MA et al., “Clustal W and Clustal tool.” J. Mol. Biol. 215:403-410 (1990). These contents and disclosures in their entirety are incorporated herein by reference.

[0054] The terms “percent complementarity” or “percent complementary” as used herein with respect to two nucleotide sequences are similar to the concept of percent identity, but refer to the percentage of nucleotides in the query sequence that best base-pair or hybridize with the nucleotides in the target sequence when the query sequence and target sequence are arranged linearly and best base-pair without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity may be between two DNA strands, two RNA strands, or between a DNA strand and an RNA strand. “Percent complementarity” is calculated by (i) best base-pairing or hybridizing the two nucleotide sequences across a comparison window in a linear and fully unfolded arrangement (i.e., without folding or secondary structures), (ii) determining the number of base-pairing positions between the two sequences across the comparison window to find the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to find the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined based on known pairings of nucleotide bases such as GC, AT, and AU via hydrogen bonds. When "percent complementarity" is calculated in relation to a reference sequence without specifying a particular comparison window, percentage identity is determined by dividing the complementary positions between the two linear sequences by the total length of the reference sequence. Therefore, in this application, when the two sequences (query and target) are optimally base-paired (allowing for mismatched or non-base-paired nucleotides), the "percent complementarity" of the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the total length of the query sequence, multiplied by 100%.

[0055] The term “operatably linked” refers to a functional relationship between a promoter or other regulatory element and the coding sequence of a relevant transcriptable DNA sequence or gene (or transgene), such that, in at least one specific tissue, developmental stage, and / or condition, the promoter or other regulatory element acts to initiate, assist, influence, induce, and / or enhance the transcription and expression of the relevant transcriptable DNA sequence or coding sequence. Regulatory elements include, but are not limited to, enhancers, leaders, transcription start sites (TSS), linkers, 5' and 3' untranslated regions (UTR), introns, polyadenylation signals, and termination regions or sequences, which are preferred, necessary, or desirable for regulating or enabling the expression of a gene or transcriptable DNA sequence in a cell. Such additional regulatory elements may be optional and can be used to promote or optimize the expression of a gene or transcriptable DNA sequence. In this application, “enhancer” can be distinguished from “promoter” in that an enhancer typically lacks a transcription start site, TATA box, or homogeneous sequence and is therefore insufficient to drive transcription on its own. As used herein, “leader” can generally be defined as the 5'-UTR DNA sequence of a gene (or transgene) located between the transcription start site (TSS) and the 5' end of the transcriptionable DNA sequence or the protein-coding sequence start site of the gene.

[0056] As is commonly understood in the art, the term “promoter” refers to a DNA sequence containing an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, which supports or enhances the transcription and expression of an associated transcriptable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include chimeric promoters, which include combinations of two or more heterologous sequences. Thus, promoters of this application may include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences (plural) known or provided herein. Promoters can be classified, for example, constitutive, developmental, tissue-specific, inducible, etc., according to various criteria relating to the expression pattern of the associated coding sequence or transcriptable sequence or gene (including transgene) operably ligated to the promoter. A promoter that drives expression in all or most tissues of a plant is called a “constitutive” promoter. A promoter that drives expression during a specific period or stage of development is called a “developmental” promoter. Promoter that drives enhanced expression in a particular plant tissue compared to other plant tissues is called a “tissue-favorable” promoter or “tissue-preferential” promoter. Therefore, a “tissue-preferential” promoter causes relatively higher or selective expression in a specific plant tissue(s) but lower levels of expression in other plant tissues(s). Promoter that is expressed in a specific plant tissue(s) but is little to no expression in other plant tissues is called a “tissue-specific” promoter. “Inducible” promoters are those that initiate transcription in response to environmental stimuli such as cold, drought, or light, or other stimuli such as wounds or chemical application. Promoter classifications can also be categorized in terms of their origin, for example, heterogeneous, homologous, chimeric, or synthetic.A “heterogeneous” promoter is a promoter sequence that has a different origin from the associated transcriptable sequence, coding sequence, or gene (or transgene) and / or is not naturally present in the plant species being transformed.

[0057] Examples of promoters that may be used herein include, but are not limited to, U.S. Patent No. 6,437,217 (Maize RS81 Promoter), U.S. Patent No. 5,641,876 (Comeactin Promoter), U.S. Patent No. 6,426,446 (Maize RS324 Promoter), U.S. Patent No. 6,429,362 (Maize PR-1 Promoter), U.S. Patent No. 6,232,526 (Maize A3 Promoter), U.S. Patent No. 6,177,611 (Constitutive Maize Promoter), U.S. Patent No. 5,322,938, No. 5,352,605, No. 5,359,142, and No. 5,530,196 (35S Promoter). Examples include U.S. Patent No. 6,433,252 (Maize L3 Oleosin Promoter), U.S. Patent No. 6,429,357 (Comeactin 2 Promoter and Comeactin 2 Intron), U.S. Patent No. 5,837,848 (Root-Specific Promoter), U.S. Patent No. 6,294,714 (Photoinducible Promoter), U.S. Patent No. 6,140,078 (Salt-Inducible Promoter), U.S. Patent No. 6,252,138 (Pathogen-Inducible Promoter), U.S. Patent No. 6,175,060 (Phosphorus Deficiency-Inducible Promoter), U.S. Patent No. 6,635,806 (Gamma-Coixin Promoter), and U.S. Patent Application No. 09 / 757,089 (Maize Chloroplast Aldolase Promoter).Additional promoters that can be used include the nopalin synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (carried by the tumor induction plasmid of Agrobacterium tumefaciens), the Kalimovirus promoter, e.g., the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9:315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313:810-812), the sesame mosaic virus 35S promoter (US Patent Nos. 6,051,753 and 5,378,619), and the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences). These include the R gene complex promoter (Sciences, USA (1990) 87:4144-4148), the chlorophyll a / b binding protein gene promoter, PC1SV (US Patent No. 5,850,019), and the AGRtu.nos promoter (GenBank registration number V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1:561-573; Bevan et al., 1983).

[0058] Promoter hybrids can be constructed to promote transcriptional activity (see U.S. Patent No. 5,106,739), or to combine desired transcriptional activity, inducibility, and tissue-specific or developmental specificity. Promoter functional in plants includes, but is not limited to, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatio-temporally regulated promoters. Other tissue-promoting, tissue-specific, or developmentally regulated promoters are also known in the art and are expected to be useful in the practice of this disclosure.

[0059] As used herein, “heterogeneous” with respect to a promoter means a promoter sequence that has a different origin from the associated transcriptable DNA sequence, coding sequence, or gene (or transgene) and / or is not naturally present in the plant species being transformed. More broadly, the term “heterogeneous” refers to a combination of two or more DNA molecules or sequences, such as a promoter and an associated transcriptable DNA sequence, coding sequence, or gene, that is artificial and not normally found in nature.

[0060] Furthermore, the term “recombinant” in relation to polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc. refers to polynucleotide or protein molecules or sequences that are artificial and not normally found in nature, and / or exist in circumstances not normally found in nature, and includes polynucleotide (DNA or RNA) molecules, proteins, constructs, etc. that include combinations of polynucleotide or protein sequences that would not naturally exist consecutively or in close proximity to each other without human intervention, and / or polynucleotide molecules, proteins, constructs, etc. that include at least two polynucleotide or protein sequences that are heterogeneous to each other. Recombinant polynucleotide or protein molecules, constructs, etc. may include polynucleotide or protein sequences that (i) are separated from other polynucleotide or protein sequences that exist in close proximity to each other in nature, and / or (ii) are adjacent to (or consecutive to) other polynucleotide or protein sequences that do not naturally exist in close proximity to each other. Such recombinant polynucleotide molecules, proteins, constructs, etc. may also refer to genetically modified and / or extracellularly constructed polynucleotide or protein molecules or sequences. For example, recombinant DNA molecules can include any suitable plasmid, vector, etc., and may include linear or circular DNA molecules. Such plasmids, vectors, etc. may contain various maintenance elements, including prokaryotic origins of replication and selection markers, as well as one or more transgenes or expression cassettes, in addition to possibly plant selection marker genes.

[0061] As used herein, “adjacent” refers to a nucleic acid sequence that is in the vicinity or adjacent to another nucleic acid sequence. In one embodiment, adjacent nucleic acid sequences are physically linked. In another embodiment, adjacent nucleic acid sequences or genes are located immediately next to each other such that there are no interposing nucleotides between the endpoint of the first nucleic acid sequence and the starting point of the second nucleic acid sequence. In one embodiment, the second gene used in the first gene is adjacent to each other if they are separated by fewer than 50,000, 25,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,500, 2,000, 1,750, 1,500, 1,250, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 20, 10, 5, 4, 3, 2, or 1 nucleotide.

[0062] In one embodiment, the methods and compositions provided herein include a vector. As used herein, the terms “vector” and “plasmid” are used synonymously and refer to a circular double-stranded DNA molecule physically separated from chromosomal DNA. In one embodiment, the plasmid or vector used herein can be replicated in vivo. As used herein, “transformation vector” is a plasmid capable of transforming plant cells. In one embodiment, the plasmid provided herein is a bacterial plasmid. In another embodiment, the plasmid provided herein is an Agrobacterium Ti plasmid or derived from an Agrobacterium Ti plasmid.

[0063] In one embodiment, the plasmids or vectors provided herein are recombinant vectors. As used herein, the term “recombinant vector” refers to a vector formed by experimental methods of genetic recombination, such as molecular cloning. In another embodiment, the plasmids provided herein are synthetic plasmids. As used herein, “synthetic plasmid” is an artificially created plasmid capable of performing the same function (e.g., replication) as a natural plasmid (e.g., a Ti plasmid). Those skilled in the art can create novel synthetic plasmids by synthesizing plasmids from individual nucleotides or by splicing nucleic acid molecules together from different existing plasmids.

[0064] As used herein, “modified” refers to a state in which plants, seeds, plant components, plant cells, and plant genomes have been altered or modified from their natural or native state. For example, the “native transcript” of a gene refers to the RNA transcript produced from an unmodified gene. Typically, native transcripts are sense transcripts. Modified plants or seeds have molecular changes in their genetic material, such as genetic or epigenetic modifications. Typically, modified plants or seeds, or their parent or precursor lines, have been subjected to mutagenesis, genome editing (e.g., by methods using site-specific nucleases, but not limited to these), genetic transformation (e.g., by Agrobacterium transformation or by methods using a microparticle gun, but not limited to these), or a combination thereof. In one embodiment, the modified plants provided herein do not contain non-plant genetic material or sequences. In yet another embodiment, the modified plants provided herein do not contain interspecies genetic material or sequences. In one aspect, the disclosure provides methods and compositions relating to modified plants, seeds, plant components, plant cells, and products made from modified plants, seeds, plant parts, and plant cells. In one aspect, the modified seeds provided herein give rise to the modified plants provided herein. In one aspect, the modified plants, seeds, plant components, plant cells, or plant genomes provided herein include the recombinant DNA constructs or vectors provided herein. In another aspect, the products provided herein include the modified plants, plant components, plant cells, or plant chromosomes or genomes provided herein.This disclosure provides modified plants having desirable or enhanced properties, for example, but not limited to, resistance to diseases, insects, or pests (e.g., viral resistance, bacterial resistance, fungal resistance, nematode resistance, arthropod resistance, gastropod resistance); herbicide resistance; environmental stress resistance; improved quality, for example, yield, nutritional enhancement, environmental or stress tolerance; any desirable changes in the physiology, growth, development, morphology or plant products, including starch production, modified oil production, high oil production, modified fatty acid content, high protein production, fruit ripening, animal and human nutrition enhancement, biomacromolecule production, pharmaceutically and secretably produced peptides; improved processing properties; improved digestibility; low raffinose; industrial enzyme production; improved flavor; nitrogen fixation; hybrid seed production; and fiber production.

[0065] As used herein, “genome editing” or “editing” refers to targeted mutagenesis, insertion, deletion, inversion, substitution, or translocation of a nucleotide sequence of interest in the genome using targeted editing techniques. The nucleotide sequence of interest may be of any length, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 5000, at least 10,000, or at least 25,000 nucleotides. As used herein, “targeted editing techniques” refers to any method, protocol, or technique that enables precise and / or targeted editing of a specific location in the genome (e.g., editing is not random). The use of site-specific nucleases is an example of targeted editing techniques, but is not limited to these. Another non-limiting example of targeted editing techniques is the use of one or more tether-guided oligos (tgOligo). As used herein, “targeted editing” means a targeted mutagenesis, insertion, deletion, inversion, or substitution caused by a targeted editing technique. The nucleotide sequence of interest may be an endogenous genomic sequence or a transgenic sequence.

[0066] In one embodiment, “targeted editing technology” refers to any method, protocol, or technique that enables precise and / or targeted editing of specific locations within the genome (e.g., editing is not random). The use of site-specific nucleases is an example of targeted editing technology, but is not limited to this.

[0067] In one embodiment, an endogenous locus or endogenous gene is edited using targeted editing techniques. In another embodiment, a transgene is edited using targeted editing techniques. As used herein, “endogenous gene” or “native copy” of a gene means a gene that originates within a given organism, cell, tissue, genome, or chromosome. An “endogenous gene” or “native copy” of a gene is a gene that has not been previously modified by human action.

[0068] As used herein, “locus” refers to a specific location on a chromosome or other nucleic acid molecule. A locus may include, but is not limited to, polynucleotides that code for proteins or RNA. A locus may also include non-coding RNA. A locus may include genes. A locus may include promoters, 5' untranslated regions (UTRs), exons, introns, 3'-UTRs, or any combination thereof. A locus may include coding regions.

[0069] As used herein, “physically linked” means two or more loci located on the same nucleic acid molecule.

[0070] As used herein, “coding region,” “gene region,” or “gene” refers to a polynucleotide capable of producing a functional unit (e.g., a protein or a non-coding RNA molecule). A “coding region,” “gene,” or “gene region” may include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription termination site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. A “coding region sequence,” “gene sequence,” or “gene region sequence” may include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription termination site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. In one embodiment, a “gene” encodes a non-coding RNA molecule or its precursor. In another embodiment, a “gene” encodes a protein.

[0071] Non-coding RNA molecules include, but are not limited to, microRNAs (miRNAs), miRNA precursors (premiRNAs), small interfering RNAs (siRNAs), small RNAs (18-26 nt in length) and their encoding precursors, heterochromatin siRNAs (hc-siRNAs), Piwi-interacting RNAs (piRNAs), hairpin double-stranded RNAs (hairpin dsRNAs), trans-acting siRNAs (ta-siRNAs), naturally occurring antisense siRNAs (nat-siRNAs), CRISPR RNAs (crRNAs), tracer RNAs (tracrRNAs), guide RNAs (gRNAs), and single guide RNAs (sgRNAs). In one embodiment, the non-coding RNAs provided herein are selected from the group consisting of microRNAs, small interfering RNAs, secondary small interfering RNAs, transfer RNAs, ribosomal RNAs, trans-acting small interfering RNAs, naturally occurring antisense small interfering RNAs, heterochromatin small interfering RNAs, and their precursors. In another embodiment, the non-coding RNAs provided herein are selected from the group consisting of miRNA, pre-miRNA, siRNA, hc-siRNA, piRNA, hairpin dsRNA, ta-siRNA, nat-siRNA, crRNA, tracrRNA, gRNA, and sgRNA. The non-coding RNAs are often 100% complementary to the non-coding RNA target site, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% complementary to the non-coding RNA target site. The non-coding RNA target site may be located on a DNA or RNA molecule. Hybridization (or binding) of non-coding RNA to a non-coding RNA target site can result in a variety of outcomes. For example, some non-coding RNAs (but not limited to, miRNAs, siRNAs, ta-siRNAs) assist in cleaving mRNA transcripts containing complementary non-coding RNA target sites.Alternatively, non-coding RNAs (but not limited to miRNAs, siRNAs, ta-siRNAs) may assist in inhibiting protein translation of mRNA transcripts by binding to non-coding RNA target sites on mRNA. Some non-coding RNAs (but not limited to hc-siRNAs) assist in inducing epigenetic changes in DNA. In one embodiment, the non-coding RNA target site is a miRNA target site or an siRNA target site. In another embodiment, the gene provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten non-coding RNA target sites. In yet another embodiment, the gene provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heterogeneous non-coding RNA target sites. In another embodiment, the dominant-negative alleles provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten non-coding RNA target sites. In another embodiment, the dominant-negative alleles provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heterologous non-coding RNA target sites. In one embodiment, the endogenous gene provided herein is modified to include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heterologous non-coding RNA target sites.

[0072] As used herein, "allele" refers to a given locus or gene variant within the genome. A cell is considered homozygous for a given locus if the same allele is present on both chromosomes of a chromosome pair within that cell. A cell is heterozygous for a given locus if each member of a chromosome pair contains a different allele for that locus. While at least one allele is possible for a given locus, typically, multiple alleles are possible for any given locus within the genome.

[0073] As used herein, the terms “suppress,” “inhibit,” “inhibit,” “to inhibit,” and “downregulate” are defined as any method known in the art or described herein that reduces the expression or function of a gene product (e.g., mRNA, protein, non-coding RNA). “Inhibition” may relate to a comparison of two cells, e.g., a modified cell and a control cell. Inhibition of the expression or function of a gene product may also relate to a comparison in plant cells, organelles, organs, tissues, or plant components within the same plant or between different plants, and include comparisons at developmental or temporal stages within the same plant or plant component or between plants or plant components. “Inhibition” includes any relative reduction in the function or production of the gene product of interest, and includes the complete cessation of the function or production of that gene product. The term “inhibition” encompasses any method or composition that downregulates the translation and / or transcription of a target gene product or the functional activity of a target gene product. “Inhibition” does not necessarily include the complete cessation of the expression of a gene product. In one embodiment, the gene product in the modified cells provided herein includes an expression that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lower than the expression of the gene product in the control cells. In another embodiment, the gene products in the modified cells provided herein include expression levels that are 1% to 100%, 1% to 95%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 25%, 5% to 50%, 5% to 75%, 5% to 100%, 10% to 25%, 10% to 50%, 10% to 75%, 10% to 100%, 25% to 50%, 25% to 75%, 25% to 100%, or 50% to 100% lower than the expression levels of the gene products in the control cells.

[0074] As used herein, “target site” refers to a location in a polynucleotide sequence where a site-specific nuclease binds and cleaves, introducing a double-strand break into the nucleic acid backbone. In another embodiment, the target site comprises a sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 nucleotides. In another embodiment, the target site provided herein comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides. In one embodiment, a site-specific nuclease binds to a target site. In another embodiment, a site-specific nuclease binds to a target site via an inducible non-coding RNA (i.e., CRISPR RNA or a single guide RNA, etc., both described in detail below). In one embodiment, the non-coding RNA provided herein is complementary to the target site. It will be understood that complete complementarity is not required for the non-coding RNA to bind to the target site. At least one, at least two, at least three, at least four, or at least five, at least six, at least seven, or at least eight mismatches between the target site and the non-coding RNA may be acceptable. As used herein, “target region” or “targeting region” refers to the polynucleotide sequence that is to be modified. In one embodiment, the “target region,” “targeting region,” or “target gene” is flanked to two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more target sites. A "target gene" refers to a polynucleotide sequence that codes for a gene that is to be modified.In one embodiment, a polynucleotide sequence containing a target gene further comprises one or more target sites. In another embodiment, a target region comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more target genes. In one embodiment, but not limited to, a target region may be subject to deletion or inversion. As used herein, “flanked” means two or more target sites that physically surround a target region, with one target site located on each side of the target region.

[0075] The target site may be located within the polynucleotide sequence encoding the leader, enhancer, transcription start site, promoter, 5'-UTR, exon, intron, 3'-UTR, polyadenylation site, or termination sequence. As will be understood, the target site may also be located upstream or downstream of the sequence encoding the leader, enhancer, transcription start site, promoter, 5'-UTR, exon, intron, 3'-UTR, polyadenylation site, or termination sequence. In one aspect, the target site is located within 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 300, 400, 2500, 600, 700, 800, 900, 1000, 1250, 12500, 12500, 12500, 12500, 12500, 12500, 12500,

[0076] As used herein, “upstream” refers to the nucleic acid sequence located before the 5' end of a ligated nucleic acid sequence. As used herein, “downstream” refers to the nucleic acid sequence located after the 3' end of a ligated nucleic acid sequence. As used herein, “5'” refers to the starting point of a coding DNA sequence or RNA molecule. As used herein, “3'” refers to the end of a coding DNA sequence or RNA molecule. As will be understood, “inversion” refers to reversing the orientation of a given polynucleotide sequence. For example, if the sample sequence 5'-ATGATC-3' is inverted, it becomes the reversed 5'-CTAGTA-3'. Furthermore, the sample sequence 5'-ATGATC-3' is considered to be in the “opposite orientation” relative to the sample sequence 5'-CTAGTA-3'.

[0077] As used herein, “donor molecule” is defined as a nucleic acid sequence selected for site-directed, targeted insertion into a genome. In one embodiment, the donor molecule includes a “donor sequence.” In one embodiment, the targeted editing techniques provided herein include the use of one or more, two or more, three or more, four or more, or five or more donor molecules or donor sequences. The donor molecules or donor sequences provided herein may be of any length. For example, the donor molecules or donor sequences provided herein have nucleotide lengths between 2 and 50,000, between 2 and 10,000, between 2 and 5,000, between 2 and 1,000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1,000, between 15 and 5,000, between 18 and 30, between 18 and 26, between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 500, between 20 and 1,000, between 20 and 5,000, or between 20 and 10,000. The donor molecules or donor sequences may include one or more genes encoding gene sequences that are actively transcribed and / or translated. Such transcribed sequences may encode proteins or non-coding RNAs. In one embodiment, the donor molecule or donor sequence may contain a polynucleotide sequence that does not contain a functional gene or an entire gene (i.e., the donor molecule may simply contain a regulatory sequence such as a promoter), or it may not contain any identifiable gene expression elements or any actively transcribed gene sequences. Furthermore, the donor molecule or donor sequence may be linear or circular, and single-stranded or double-stranded. It may be delivered to cells as a naked nucleic acid, as a complex with one or more delivery agents (e.g., liposomes, poloxamers, protein-encapsulated T chains, etc.), or contained in a bacterial or viral delivery vehicle, such as Agrobacterium tumefaciens or geminivirus, respectively. In another embodiment, the donor molecule or donor sequence provided herein is operably linked to a promoter.In further embodiments, the donor molecule or donor sequence provided herein is transcribed into RNA. In another embodiment, the donor molecule or donor sequence provided herein is not operably ligated to a promoter.

[0078] In some embodiments, the donor molecules or donor sequences provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes. In some embodiments, the donor molecules or donor sequences provided herein may comprise no genes. Genes provided herein may include, but are not limited to, insecticide resistance genes, herbicide resistance genes, nitrogen use efficiency genes, water use efficiency genes, nutritional quality genes, DNA binding genes, selection marker genes, RNAi constructs, site-directed genome modification enzyme genes, single guide RNAs for CRISPR / Cas9 systems, geminivirus-based expression cassettes, or plant virus expression vector systems. In one embodiment, the donor molecule or donor sequence comprises a polynucleotide encoding a promoter. In another embodiment, the donor molecules or donor sequences provided herein comprises a polynucleotide encoding a tissue-specific or tissue-preferential promoter. In yet another embodiment, the donor molecules or donor sequences provided herein comprise a polynucleotide encoding a constitutive promoter. In another embodiment, the donor molecule or donor sequence provided herein comprises a polynucleotide encoding an inducible promoter. In another embodiment, the donor molecule or donor sequence comprises a polynucleotide encoding a structure selected from the group consisting of a leader, enhancer, transcription start site, 5'-UTR, exon, intron, 3'-UTR, polyadenylation site, transcription termination site, promoter, full-length gene, partial gene, gene, or non-coding RNA. In one embodiment, the donor molecule or donor sequence provided herein comprises one or more, two or more, three or more, four or more, or five or more designed elements.

[0079] As used herein, “donor template” may be a recombinant DNA donor template and is defined as a nucleic acid molecule having a nucleic acid template or insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell by repair of nicks or double-strand DNA breaks in the genome of a plant cell. For example, a “donor template” may be used as a template for site-directed integration of a DNA segment encoding an antisense sequence of interest, or for introducing mutations such as insertions or deletions into a target site in the genome of a plant. Targeted genome editing techniques provided herein may involve the use of one or more, two or more, three or more, four or more, or five or more donor templates. A “donor template” may be a single-stranded or double-stranded DNA molecule, an RNA molecule, or a plasmid. The “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, and it may be of any preferred length. For example, the insertion sequences for the donor mold are between 2 and 50,000, between 2 and 10,000, between 2 and 5,000, between 2 and 1,000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1,000, between 15 and 5,000, between 18 and 30, between 18 and 26, between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 500, and between 20 and 500. The length of a nucleotide or base pair can be between 1,000, between 20 and 5,000, between 20 and 10,000, between 50 and 250, between 50 and 500, between 50 and 1,000, between 50 and 5,000, between 50 and 10,000, between 100 and 250, between 100 and 500, between 100 and 1,000, between 100 and 5,000, between 250 and 500, between 250 and 1,000, between 250 and 5,000, or between 250 and 10,000. The donor template also has at least one homologous sequence or homologous arm (e.g., two homologous arms) that directs the integration of a mutation or insertion sequence into a target site in the plant genome via homologous recombination, wherein the homologous sequence or homologous arm(s) are identical or complementary, or have percent identity or percent complementarity to a sequence at or near the target site in the plant genome.If the donor mold contains homologous arms and inserts, the homologous arms may flank or surround the inserts of the donor mold.

[0080] The donor template may be linear or circular, and may be single-stranded or double-stranded. The donor template may be delivered to cells as naked nucleic acid (e.g., via a particle gun), as a complex with one or more delivery agents (e.g., liposomes, proteins, poloxamers, protein-encapsulated T chains, etc.), or contained in a bacterial or viral delivery vehicle, such as Agrobacterium tumefaciens or geminivirus, respectively. The insertion sequence of the donor template or the insertion sequences provided herein may include a transcribed DNA sequence or segment that can be transcribed to all or part of an RNA molecule, such as an antisense sequence or portion of an RNA molecule.

[0081] As used herein, “designed element” refers to a polynucleotide capable of inducing a desired expression pattern of operablely linked polynucleotides. In one embodiment, the designed element contains at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, or at least 5000 nucleotides. In another embodiment, the designed element contains 20 to 50 nucleotides. In yet another embodiment, the designed element contains 10-5000, 10-2500, 10-1000, 10-500, 10-100, 20-50, 20-100, 20-500, 20-1000, 20-2000, 20-5000, 50-100, 50-500, 50-100, 50-1000, or 50-5000 nucleotides. In one embodiment, the designed element contains a constitutive promoter. In another embodiment, the designed element contains an inducible promoter. In another embodiment, the designed element contains a tissue-specific or tissue-preferential promoter. In another embodiment, the designed element contains a native promoter. In another embodiment, the designed element contains a non-native promoter. In another embodiment, the designed element contains a tissue-specific or tissue-preferential promoter element. In another embodiment, the designed element contains a transcription enhancer element. In another embodiment, the designed element includes a transfer repressor element.

[0082] One aspect of this application relates to a method for screening and selecting cells for targeted editing, and a method for selecting cells that include targeted editing. Nucleic acids can be isolated using a variety of techniques. For example, nucleic acids can be isolated using any method, including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The degree of purity of the polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0083] In one embodiment, this disclosure provides a method for detecting recombinant nucleic acids and polypeptides in modified and unmodified plant cells. Nucleic acids can also be detected using hybridization, though not limited to this method. Nucleic acid hybridization is described in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0084] Antibodies may be used to detect polypeptides. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal antibodies. Antibodies having a specific binding affinity to the polypeptides provided herein can be produced using methods well known in the art. The antibodies provided herein can be attached to a solid support, such as a microtiter plate, using methods known in the art.

[0085] Detection (e.g., detection of amplification products, hybridization complexes, or polypeptides) can be achieved using detectable labels. The term "label" is intended to encompass the use of both direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances.

[0086] The screening and selection of modified, manipulated, or transgenic plants or plant cells can be carried out by any methodology known to those skilled in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for the detection of polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for the detection of RNA transcripts, Sanger sequencing, next-generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for the detection of enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-conjugated immunoassays for the detection of polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are publicly known.

[0087] Genome editing or targeted editing can be performed using one or more site-specific nucleases. Site-specific nucleases can induce double-strand breaks (DSBs) at target sites in the genome sequence, which are then repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ), both natural processes. Sequence modifications such as insertions and deletions can occur at the DSB site via NHEJ repair. If two DSBs are created flanking one target region, the breaks can be repaired via NHEJ by reversing the orientation of the targeted DNA (also called "inversion"). HR can be used to incorporate a donor nucleic acid sequence into the target site. In order to incorporate a donor nucleic acid sequence (or donor molecule) into a DSB, the donor molecule contains the polynucleotide of interest flanked into first and second homologous regions, where the first and second homologous regions are homologous to each side of the DSB at the target site. Next, the intracellular homologous recombination mechanism repairs the DSB by incorporating the donor molecule into the target site. In one embodiment, the double-strand break provided herein is repaired by NHEJ. In another embodiment, the double-strand break provided herein is repaired by HR.

[0088] Although double-strand breaks occur only between two nucleotides on each strand, a double-strand break site may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least seventy, at least seventy, at least eighty, at least ninety, or at least one hundred nucleotides. As used herein, “double-strand break site” refers to a polynucleotide sequence that is recognized and bound by a site-specific nuclease or guide RNA.

[0089] In one embodiment, the vector or construct provided herein comprises a polynucleotide encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. In another embodiment, the cells provided herein already contain site-specific nucleases. In one embodiment, the polynucleotide encoding site-specific nucleases provided herein is stably transformed into cells. In another embodiment, the polynucleotide encoding site-specific nucleases provided herein is transiently transformed into cells. In another embodiment, the polynucleotide encoding site-specific nucleases is under the control of a moduloable promoter, a constitutive promoter, a tissue-specific promoter, or any promoter useful for the expression of site-specific nucleases.

[0090] In one embodiment, the vector comprises a cassette encoding a site-specific nuclease and a donor molecule in cis configuration, such that upon contact with the cell's genome, the site-specific nuclease enables site-specific integration of the donor molecule. In another embodiment, the first vector comprises a cassette encoding a site-specific nuclease and the second vector comprises a donor molecule, such that upon contact with the cell's genome, the site-specific nuclease provided in trans configuration enables site-specific integration of the donor molecule.

[0091] Site-specific nucleases provided herein can be used as part of targeted editing techniques. Non-exclusive examples of site-specific nucleases used in methods and / or compositions provided herein include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA guide nucleases (e.g., Cas9 and Cpf1), recombinases (but not limited to, e.g., serine recombinases bound to DNA recognition motifs, tyrosine recombinases bound to DNA recognition motifs), transposases (but not limited to, e.g., DNA transposases bound to DNA-binding domains), or any combination thereof. In one embodiment, the method provided herein involves the use of one or more site-specific nucleases to induce one, two, three, four, five, or six or more DSBs at one, two, three, four, five, or six or more target sites.

[0092] In one embodiment, a site-specific nuclease protein is provided to a cell. In another embodiment, a nucleic acid sequence (e.g., a vector) encoding the site-specific nuclease protein is provided to a cell. In yet another embodiment, the site-specific nuclease protein and guide RNA are provided to the cell separately. In yet another embodiment, the site-specific nuclease protein and guide RNA are provided to the cell as a complex. In one embodiment, the site-specific nuclease protein and guide RNA are assembled into a complex in vitro, in vivo, or ex vivo.

[0093] In one embodiment, the genome editing systems provided herein (e.g., meganucleases, ZFNs, TALENs, CRISPR / Cas9 systems, CRISPR / Cpf1 systems, recombinases, transposases), or combinations of genome editing systems provided herein, are used in a method for generating a dominant-negative or dominant-positive allele by introducing one or more insertions, deletions, substitutions, or inversions into intracellular loci.

[0094] Site-specific nucleases, such as meganucleases, ZFNs, TALENs, Argonaut proteins (non-specific examples of Argonaut proteins include Thermus thermophilus Argonaut (TtAgo), Pyrococcus furiosus Argonaut (PfAgo), Natronobacterium gregoryi Argonaut (NgAgo), their homologs, or their variants), Cas9 nucleases (non-specific examples of RNA guide nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4) These genes (including Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs, or their variants) induce double-strand DNA breaks at target sites in the genome sequence, which are then repaired by the innate processes of HR or NHEJ. Sequence modifications then occur at the break site. Sequence modifications may include inversions, deletions, or insertions, resulting in gene disruption in the case of NHEJ, or incorporation of nucleic acid sequences in the case of HR.

[0095] In one embodiment, the site-specific nucleases provided herein are selected from the group consisting of zinc finger nucleases, meganucleases, RNA guide nucleases, TALE-nucleases, recombinases, transposases, or any combination thereof. In another embodiment, the site-specific nucleases provided herein are selected from the group consisting of Cas9 or Cpf1. In another embodiment, the site-specific nucleases provided herein are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, C The RNA guide nucleases provided herein are selected from the group consisting of sm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs, or modified versions thereof. In another embodiment, the RNA guide nucleases provided herein are selected from the group consisting of Cas9 or Cpf1. In another embodiment, the RNA guide nucleases provided herein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, C The RNA guide nuclease is selected from the group consisting of sm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs, or modified versions thereof. In another embodiment, the RNA guide nuclease is the Cas9 nuclease or its homolog or modified version.In one embodiment, the RNA guide nuclease is the Cas9 protein or a modified version thereof derived from Streptococcus pyogenes, Streptococcus thermophilius, Staphylococcus aureus, Neisseria meningitides, or Treponema denticola. In another embodiment, the RNA guide nuclease is Cpf1 or its homolog or a modified version thereof.

[0096] In another embodiment, the methods and / or compositions provided herein comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. In yet another embodiment, the methods and / or compositions provided herein comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten polynucleotides encoding site-specific nucleases.

[0097] In one embodiment, the targeted editing technique described herein involves the use of a recombinase. In one embodiment, the tyrosine recombinase bound to the DNA recognition motif provided herein is selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one embodiment, the Cre recombinase or Gin recombinase provided herein is ligated to a zinc finger DNA binding domain. The Flp-FRT site-directed recombination system is derived from a 2μ plasmid of the baker's yeast Saccharomyces cerevisiae. In this system, Flp recombinase (flippase) rearranges sequences between flippase-recognition target (FRT) sites. The FRT site contains 34 nucleotides. Flp binds to the "arms" of the FRT site (one arm is reversed) and cleaves the FRT site at both ends of the intervening nucleic acid sequence. After cleavage, Flp rearranges the nucleic acid sequence between the two FRT sites. Cre-lox is a site-directed recombination system derived from bacteriophage P1, similar to the Flp-FRT recombination system. Cre-lox can be used for nucleic acid sequence inversions, deletions, or translocations. In this system, Cre recombinase recombinates a pair of lox nucleic acid sequences. The lox site contains 34 nucleotides, with the first and last 13 nucleotides (arms) forming a palindromic structure. During recombination, the Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves them at the lox sites. The cleaved nucleic acids are then spliced ​​together (mutual translocation), completing the recombination. In another embodiment, the lox sites provided herein are loxP, lox 2272, loxN, lox 511, lox 5171, lox71, lox66, M2, M3, M7, or M11 sites.

[0098] In another embodiment, the serine recombinase bound to the DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In another embodiment, the DNA transposase bound to the DNA binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0099] In one embodiment, the targeted editing technique described herein involves the use of a zinc finger nuclease (ZFN). A ZFN is a synthetic protein comprising an engineered zinc finger DNA-binding domain fused to the cleavage domain of a FokI restriction nuclease. ZFNs can be designed to cleave virtually any long segment of double-stranded DNA for modification of the zinc finger DNA-binding domain. A ZFN dimers from monomers consisting of nonspecific DNA-cleavage domains of a FokI nuclease fused to a zinc finger array engineered to bind to a target DNA sequence.

[0100] The DNA-binding domain of a ZFN typically consists of a 3-4 zinc finger array. Amino acids at positions -1, +2, +3, and +6 relative to the starting point of the zinc finger ∞-helix, which contribute to site-specific binding to target DNA, can be modified and customized to fit specific target sequences. Other amino acids form a consensus backbone, generating ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

[0101] Since the FokI nuclease domain requires dimerization to cleave DNA, two ZFNs with C-terminal regions are needed to join the opposing DNA strands (5–7 nt apart) at the cleavage site. If the two ZF binding sites are in a batch structure, the ZFN monomer can cleave the target site. As used herein, the term ZFN is broad and includes monomeric ZFNs capable of cleaving double-stranded DNA without assistance from another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site.

[0102] While not limited to any particular scientific theory, the DNA binding specificity of zinc finger domains can, in principle, be remanufactured using one of several methods, making it theoretically possible to construct ZFNs customized to target virtually any gene sequence. Publicly available methods for manipulating zinc finger domains include context-dependent assembly (CoDA), oligomerization pooling (OPEN), and modular assembly.

[0103] In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more ZFNs. In another embodiment, the ZFNs provided herein are capable of generating targeted DSBs. In one embodiment, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs is provided to cells by transformation methods known in the art (e.g., but not limited to, viral transfection, particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0104] In one embodiment, the targeted editing techniques described herein involve the use of meganucleases. Meganucleases are unique enzymes commonly identified in microorganisms that possess high activity and long recognition sequences (>14 nt) resulting in site-specific digestion of target DNA. Manipulated forms of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14–40 nt). Because the DNA recognition and cleavage functions of meganucleases are intertwined within a single domain, manipulating meganucleases can be more challenging than manipulating ZFNs and TALENs. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.

[0105] In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more meganucleases. In another embodiment, the meganucleases provided herein are capable of generating targeted DSBs. In one embodiment, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases is provided to cells by transformation methods known in the art (e.g., but not limited to, viral transfection, particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0106] In one embodiment, the targeted editing techniques described herein involve the use of a transcription activator-like effector nuclease (TALEN). TALEN is an artificial restriction enzyme produced by fusing a transcription activator-like effector (TALE) DNA-binding domain to a FokI nuclease domain. When each member of the TALEN pair binds to the DNA site to be flanked at the target site, the FokI monomer dimerizes, causing a double-strand DNA break at the target site. In addition to the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer and requires two constructs with unique DNA-binding domains for the site in the target genome, with appropriate orientation and spacing. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two separate TALEN binding sites, are parameters for achieving high levels of activity.

[0107] TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA-binding domain to a nuclease domain. In one embodiment, the nuclease is selected from the group consisting of PvuII, MutH, TevI, and FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. When each member of the TALEN pair binds to a DNA site to flank at the target site, the FokI monomer dimerizes, causing a double-strand DNA break at the target site.

[0108] As used herein, the term TALEN is broad and includes monomeric TALENs capable of cleaving double-stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0109] Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. X pathogens secrete TALEs into host plant cells during infection. TALEs then travel to the nucleus, where they recognize and bind to specific DNA sequences located in the promoter regions of specific genes in the host genome. TALEs have a central DNA-binding domain composed of 13 to 28 repeat monomers of 33 to 34 amino acids each. The amino acids in each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. Two variable amino acids are called repeat variable duodecimal (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and can recognize a sequence of DNA bases through the modification of the RVD. This simple relationship between amino acid sequences and DNA recognition allowed for the manipulation of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0110] Beyond the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for a site in the target genome, oriented and spaced appropriately. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two separate TALEN-binding sites, are parameters for achieving high levels of activity. The PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and its variants for use with TALE. PvuII functions as a highly specific cleavage domain when coupled to TALE (see Yank et al. 2013. PLoS One. 8:e82539). MutH can introduce specific nicks into strands in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41:e83). TevI introduces double-strand breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4:1762).

[0111] The relationship between the amino acid sequence of the TALE-binding domain and DNA recognition enables the design of proteins. TALE constructs can be designed using software programs such as DNAWorks. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., Nucleic Acids Research (2012) 40:W117-122, Cermak et al., Nucleic Acids Research (2011) 39:e82, and tale-nt.cac.cornell.edu / about.

[0112] In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more TALENs. In another embodiment, the TALENs provided herein are capable of generating targeted DSBs. In one embodiment, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs is provided to cells by transformation methods known in the art (e.g., but not limited to, viral transfection, particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0113] In one embodiment, the targeted editing techniques described herein involve the use of RNA guided nucleases. The CRISPR / Cas9 system or the CRISPR / Cpf1 system are alternatives to ZFNs and TALENs in FokI-based methods. The CRISPR system is based on an RNA guided editing nuclease that recognizes the DNA sequence at a target site using complementary base pairs.

[0114] In one embodiment, the vectors provided herein may encode any combination of nucleic acid sequences that encode an RNA guide nuclease (non-limiting examples of RNA guide nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3) The following may be included: Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs, or modified versions thereof), and optionally, guide RNA necessary to target each nuclease. As used herein, the terms “guide RNA” or “gRNA” generally refer to an RNA molecule (or collectively a group of RNA molecules) that can bind to an RNA guide endonuclease and help target the nuclease to a specific position within a target polynucleotide (e.g., DNA).

[0115] While not limited to any particular scientific theory, CRISPR / Cas nucleases are part of the adaptive immune system of bacteria and archaea, protecting them from invading nucleic acids such as viruses by sequence-dependently cleaving target DNA. Immunity is acquired by incorporating short fragments of invading DNA, known as spacers, between approximately 20-nucleotide-long CRISPR repeats at the proximal end of the CRISPR locus (CRISPR array). A well-described Cas protein is the Cas9 nuclease (also known as Csn1), part of the class 2, type II CRISPR / Cas system in Streptococcus pyogenes. See Makarova et al. Nature Reviews Microbiology (2015) doi:10.1038 / nrmicro3569. Cas9 contains a RuvC-like nuclease domain at its amino terminus and an HNH-like nuclease domain located in the middle of the protein. The Cas9 protein also contains a PAM interaction (PI) domain, a recognition lobe (REC), and a BH domain. Another type II system, the Cpf1 nuclease, acts similarly to Cas9, but Cpf1 does not require tracrRNA. See Cong et al. Science (2013) 339:819-823, Zetsche et al., Cell (2015) doi:10.1016 / j.cell.2015.09.038, U.S. Patent Publication No. 2014 / 0068797, U.S. Patent Publication No. 2014 / 0273235, U.S. Patent Publication No. 2015 / 0067922, U.S. Patent No. 8,697,359, U.S. Patent No. 8,771,945, U.S. Patent No. 8,795,965, U.S. Patent No. 8,865,406, U.S. Patent No. 8,871,445, U.S. Patent No. 8,889,356, U.S. Patent No. 8,889,418, U.S. Patent No. 8,895,308, and U.S. Patent No. 8,906,616. These are incorporated herein by reference as a whole.

[0116] When Cas9 or Cpf1 cleaves targeted DNA, an endogenous double-strand break (DSB) repair mechanism is activated. DSBs can be repaired by non-homologous end joining, which can integrate insertions or deletions (indels) into the targeted locus. If two DSBs are produced that flank a single target region, the cleavage can be repaired by reversing the orientation of the targeted DNA. Alternatively, if a donor polynucleotide homologous to the target DNA sequence is provided, the DSB can be repaired by homology-directed repair. This repair mechanism allows for the precise integration of the donor polynucleotide into the targeted DNA sequence.

[0117] While not limited to any particular scientific theory, in class 2, type II CRISPR / Cas systems, a CRISPR array containing spacers is transcribed upon encounter with recognized invasive DNA and processed into a small interfering CRISPR RNA (crRNA) approximately 40 nucleotides in length. The crRNA hybridizes with transactivating crRNA (tracrRNA) to activate the Cas9 nuclease, which guides it to the target site. The nucleic acid molecules provided herein, referred to herein as "single-stranded guide RNA (sgRNA)," can combine crRNA and tracrRNA into a single nucleic acid molecule. A prerequisite for Cas9 cleavage of the target site is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has a 5-NGG-3 sequence, but rarely a NAG sequence. Specificity is provided by a so-called "seed sequence" located approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 acts similarly to Cas9, but does not require tracrRNA. Therefore, in embodiments utilizing Cpf1, sgRNA can be replaced with crRNA. The PAM motif of Cpf1 is located upstream of the target site. Furthermore, in the case of the Cpf1 orthologues LbCpf1 and AsCpf1, the PAM sequence is 5-TTTV-3, where V can be A, C, or G. In one embodiment, when two or more sgRNAs are provided herein, the first and second sgRNAs are complementary to different strands of the double-stranded DNA molecule. In another embodiment, when two or more sgRNAs are provided herein, the first and second sgRNAs are complementary to the same strand of the double-stranded DNA molecule. As used herein, “protospacer adjacent motif” (PAM) refers to a 2-6 base pair DNA sequence located immediately upstream or downstream of the target sequence of the CRISPR complex. In another embodiment, the first and second gRNAs target different PAM sequences. In yet another embodiment, the first and second gRNAs target the same PAM sequence.

[0118] In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more Cas9 nucleases. In one embodiment, the methods and / or compositions provided herein comprise one or more polynucleotides encoding one or more, two or more, three or more, four or more, or five or more Cas9 nucleases. In another embodiment, the Cas9 nucleases provided herein are capable of generating targeted DSBs. In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more Cpf1 nucleases. In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more Cpf1 nucleases. In another embodiment, the Cpf1 nucleases provided herein are capable of generating targeted DSBs.

[0119] When the Cas9 nuclease hybridizes to a target site via sgRNA, Cas9 generates two blunt-end breaks in the double-stranded DNA. The "target strand" of the double-stranded DNA is complementary to the sgRNA, and the "non-target strand" contains a PAM motif adjacent to the break site on the non-target strand and at its 3' end. Cas9 retains the target strand and the PAM motif, but the 3' end of the non-target strand is free and is referred to as the "3' flap". In one embodiment, the 3' flap contains at least 10, at least 15, at least 20, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 nucleotides.

[0120] In one embodiment, a vector comprising a polynucleotide encoding a site-specific nuclease and optionally one or more, two or more, three or more, or four or more sgRNAs is provided to plant cells by transformation methods known in the art (e.g., particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation). In one embodiment, a vector comprising a polynucleotide encoding a Cas9 nuclease and optionally one or more, two or more, three or more, or four or more sgRNAs is provided to plant cells by transformation methods known in the art (e.g., particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation). In another embodiment, a vector comprising a polynucleotide encoding Cpf1 and optionally one or more, two or more, three or more, or four or more crRNAs is provided to cells by transformation methods known in the art (e.g., but not limited to, viral transfection, particle gun, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0121] In one embodiment, the RNA guide nucleases provided herein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, C The group is selected from mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs or modified versions thereof, Argonaut (non-exclusive examples of Argonaut proteins include Thermus thermophilus Argonaut (TtAgo), Pyrococcus furiosus Argonaut (PfAgo), Natronobacterium gregoryi Argonaut (NgAgo), their homologs, and modified versions thereof), DNA guides for Argonaut proteins, and any combination thereof. In another embodiment, the RNA guide nucleases provided herein are selected from the group consisting of Cas9 and Cpf1. The RNA guide nucleases provided herein include Cas9. In one embodiment, the RNA guide nucleases provided herein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, and Csm4. The group is selected from Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs, or modified versions thereof.In one aspect, site-specific nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, and Cm The group is selected from r1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, TtAgo, PfAgo, and NgAgo. In another aspect, RNA guide nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, C The group is selected from mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, TtAgo, PfAgo, and NgAgo.

[0122] Nucleases such as Cas9 can also be manipulated to form catalytically inactivated forms, such as catalytically inactivated Cas9 (dCas9). dCas9 binds to DNA at a target site specified by gRNA, creating a loop structure accessible by template-based editing (Figure 11, Panel 1). dCas9 may be further modified to form a fusion with an ssDNA binding domain to further facilitate template-based editing (Figure 11, Panel 2). The editing efficiency of this modified dCas9-ssDNA binding scheme is expected to be higher than that of the dCas9-alone approach because the ssDNA template binds to the dCas9 complex and is closer to the gRNA target. As used herein, “inactivated Cas nuclease” (dCas) refers to an enzymatically inactive form of Cas nuclease protein that can bind to DNA but cannot cleave it. In one embodiment, the nuclease provided herein is a dCas. In another embodiment, the site-specific nuclease provided herein is a dCas.

[0123] In one embodiment, loci in eukaryotic cells can be edited using the methods and compositions provided herein. In one embodiment, the eukaryotic cells provided herein are part of a multicellular eukaryote. In another embodiment, the eukaryotic cells provided herein are unicellular organisms. In yet another embodiment, the eukaryotic cells provided herein are selected from the group consisting of animal cells, plant cells, fungal cells, and protist cells. In one embodiment, the animal cells provided herein are selected from the group consisting of insect cells, arachnid cells, arthropod cells, crustacean cells, rotifer cells, cnidarian cells, flatworm cells, mollusk cells, gastropod cells, nematode cells, annelid cells, vertebrate cells, mammalian cells, avian cells, fish cells, reptile cells, and amphibian cells. In yet another embodiment, the plant cells provided herein are monocotyledonous plant cells or dicotyledonous plant cells. In yet another embodiment, the plant cells provided herein are algal cells. In yet another embodiment, the plant cells provided herein are selected from the group consisting of maize cells, wheat cells, sorghum cells, rapeseed cells, soybean cells, alfalfa cells, cotton cells, and rice cells.In yet another embodiment, the plant cells provided herein include acacia cells, alfalfa cells, dill cells, apple cells, apricot cells, artichoke cells, yellow radish cells, asparagus cells, avocado cells, banana cells, barley cells, bean cells, sugar beet cells, blackberry cells, blueberry cells, broccoli cells, Brussels sprout cells, cabbage cells, rapeseed cells, cantaloupe cells, carrot cells, cassava cells, and cabbage cells. Flower cells, celery cells, Chinese cabbage cells, cherry cells, coriander leaf cells, citrus cells, clementine cells, coffee cells, corn cells, cotton cells, cucumber cells, Douglas fir cells, eggplant cells, endive cells, chrysanthemum cells, eucalyptus cells, fennel cells, fig cells, forest tree cells, gourd cells, grape cells, grapefruit cells, honeydew cells, jicama cells, kiwi cells, lettuce cells, chive cells, lemon cells, lime cells, Pine cells, mango cells, maple cells, melon cells, mushroom cells, nectarine cells, nut cells, oat cells, okra cells, onion cells, orange cells, ornamental plant cells, papaya cells, parsley cells, pea cells, peach cells, peanut cells, pear cells, pepper cells, persimmon cells, pine cells, pineapple cells, plantain cells, plum cells, pomegranate cells, poplar cells, potato cells, pumpkin cells, quince cells, radiata pine cells, radicchio cells The plant cells provided herein are selected from the group consisting of corn cells, radish cells, rapeseed cells, raspberry cells, rice cells, rye cells, sorghum cells, southern pine cells, soybean cells, spinach cells, melon cells, strawberry cells, sugar beet cells, sugarcane cells, sunflower cells, sweet corn cells, sweet potato cells, sweet leaf sweet cell, tangerine cells, tea cells, tobacco cells, tomato cells, grass cells, climbing plant cells, watermelon cells, wheat cells, yam cells, and zucchini cells. In another embodiment, the plant cells provided herein are selected from the group consisting of maize cells, soybean cells, rapeseed cells, cotton cells, wheat cells, and sugarcane cells.

[0124] In yet another embodiment, the manipulated plants provided herein are algae. In yet another embodiment, the manipulated plants or seeds provided herein are selected from the group consisting of maize plants, wheat plants, sorghum plants, rapeseed plants, soybean plants, alfalfa plants, cotton plants, and rice plants. In yet another embodiment, the manipulated plants or seeds provided herein are acacia plants, alfalfa plants, dill plants, apple plants, apricot plants, artichoke plants, yellow radish plants, asparagus plants, avocado plants, banana plants, barley plants, legume plants, sugar beet plants, blackberry plants, blueberry plants, broccoli plants, Brussels sprout plants, cabbage plants, rapeseed plants, cantaloupe plants, carrot plants, cassava plants. Bamboo plants, cauliflower plants, celery plants, Chinese cabbage plants, cherry plants, coriander leaf plants, citrus plants, clementine plants, coffee plants, corn plants, cotton plants, cucumber plants, Douglas fir plants, eggplant plants, endive plants, chrysanthemum plants, eucalyptus plants, fennel plants, fig plants, forest trees, gourd plants, grape plants, grapefruit plants, honeydew plants, jicama plants, kiwi plants, lettuce plants, chive plants, lemon plants, ra Immune plants, pine plants, mango plants, maple plants, melon plants, mushroom plants, nectarine plants, nut plants, oat plants, okra plants, onion plants, orange plants, ornamental plants, papaya plants, parsley plants, pea plants, peach plants, peanut plants, pear plants, pepper plants, persimmon plants, pine plants, pineapple plants, plantain plants, plum plants, pomegranate plants, poplar plants, potato plants, pumpkin plants, quince plants, radiata pine plants, radiata The group is selected from the following: yicchio plants, radish plants, rapeseed plants, raspberry plants, rice plants, rye plants, sorghum plants, southern pine plants, soybean plants, spinach plants, melon plants, strawberry plants, sugar beet plants, sugarcane plants, sunflower plants, sweet corn plants, sweet potato plants, sweetleaf sweetgum plants, tangerine plants, tea plants, tobacco plants, tomato plants, grass plants, vines, watermelon plants, wheat plants, yam plants, and zucchini plants.In another embodiment, the plants provided herein are selected from the group consisting of maize plants, soybean plants, rapeseed plants, cotton plants, wheat plants, and sugarcane plants.

[0125] In yet another embodiment, the modified plants provided herein are algae. In yet another embodiment, the modified plants provided herein are selected from the group consisting of maize plants, wheat plants, sorghum plants, rapeseed plants, soybean plants, alfalfa plants, cotton plants, and rice plants. In yet another embodiment, the modified plants provided herein are acacia plants, alfalfa plants, dill plants, apple plants, apricot plants, artichoke plants, yellow radish plants, asparagus plants, avocado plants, banana plants, barley plants, legume plants, sugar beet plants, blackberry plants, blueberry plants, broccoli plants, Brussels sprout plants, cabbage plants, rapeseed plants, cantaloupe plants, carrot plants, and cassava plants. Cauliflower plants, celery plants, Chinese cabbage plants, cherry plants, coriander leaf plants, citrus plants, clementine plants, coffee plants, corn plants, cotton plants, cucumber plants, Douglas fir plants, eggplant plants, endive plants, chrysanthemum plants, eucalyptus plants, fennel plants, fig plants, forest trees, gourd plants, grape plants, grapefruit plants, honeydew plants, jicama plants, kiwi plants, lettuce plants, chives, lemon plants, lime Plants, pine plants, mango plants, maple plants, melon plants, mushroom plants, nectarine plants, nut plants, oat plants, okra plants, onion plants, orange plants, ornamental plants, papaya plants, parsley plants, pea plants, peach plants, peanut plants, pear plants, pepper plants, persimmon plants, pine plants, pineapple plants, plantain plants, plum plants, pomegranate plants, poplar plants, potato plants, pumpkin plants, quince plants, radiata pine plants, radi The group is selected from the following: cucumber plants, radish plants, rapeseed plants, raspberry plants, rice plants, rye plants, sorghum plants, southern pine plants, soybean plants, spinach plants, melon plants, strawberry plants, sugar beet plants, sugarcane plants, sunflower plants, sweet corn plants, sweet potato plants, sweetleaf sweetgum plants, tangerine plants, tea plants, tobacco plants, tomato plants, grass plants, vines, watermelon plants, wheat plants, yam plants, and zucchini plants.

[0126] In yet another aspect, the modified seeds provided herein are selected from the group consisting of maize seeds, wheat seeds, sorghum seeds, rapeseed seeds, soybean seeds, alfalfa seeds, cotton seeds, and rice seeds. In yet another aspect, the modified seeds provided herein are acacia seeds, alfalfa seeds, dill seeds, apple seeds, apricot seeds, artichoke seeds, yellow radish seeds, asparagus seeds, avocado seeds, banana seeds, barley seeds, legume seeds, sugar beet seeds, blackberry seeds, blueberry seeds, broccoli seeds, Brussels sprout seeds, cabbage seeds, rapeseed seeds, cantaloupe seeds, carrot seeds, cassava seeds, Cauliflower seeds, celery seeds, Chinese cabbage seeds, cherry seeds, coriander leaf seeds, citrus seeds, clementine seeds, coffee seeds, corn seeds, cotton seeds, cucumber seeds, Douglas fir seeds, eggplant seeds, endive seeds, chrysanthemum seeds, eucalyptus seeds, fennel seeds, fig seeds, forest tree seeds, gourd seeds, grape seeds, grapefruit seeds, honeydew seeds, jicama seeds, kiwi seeds, lettuce seeds, chive seeds, lemon seeds, lime seeds Pine seeds, mango seeds, maple seeds, melon seeds, mushroom seeds, nectarine seeds, nut seeds, oat seeds, okra seeds, onion seeds, orange seeds, ornamental plant seeds, papaya seeds, parsley seeds, pea seeds, peach seeds, peanut seeds, pear seeds, pepper seeds, persimmon seeds, pine seeds, pine seeds, pineapple seeds, plantain seeds, plum seeds, pomegranate seeds, poplar seeds, potato seeds, pumpkin seeds, quince seeds, radiata pine seeds, radish The seeds are selected from the group consisting of kio seeds, radish seeds, rapeseed seeds, raspberry seeds, rice seeds, rye seeds, sorghum seeds, southern pine seeds, soybean seeds, spinach seeds, melon seeds, strawberry seeds, sugar beet seeds, sugarcane seeds, sunflower seeds, sweet corn seeds, sweet potato seeds, sweet leaf styraciflua seeds, tangerine seeds, tea seeds, tobacco seeds, tomato seeds, grass seeds, climbing plant seeds, watermelon seeds, wheat seeds, yam seeds, and zucchini seeds.

[0127] In yet another embodiment, the modified chromosomes provided herein are algae. In yet another embodiment, the modified chromosomes provided herein are selected from the group consisting of maize chromosomes, wheat chromosomes, sorghum chromosomes, rapeseed chromosomes, soybean chromosomes, alfalfa chromosomes, cotton chromosomes, and rice chromosomes.In yet another embodiment, the modified chromosomes provided herein include acacia chromosome, alfalfa chromosome, dill chromosome, apple chromosome, apricot chromosome, artichoke chromosome, yellow radish chromosome, asparagus chromosome, avocado chromosome, banana chromosome, barley chromosome, legume chromosome, sugar beet chromosome, blackberry chromosome, blueberry chromosome, broccoli chromosome, Brussels sprout chromosome, cabbage chromosome, rapeseed chromosome, cantaloupe chromosome, carrot chromosome, cassava chromosome, cauliflower chromosome. - chromosome, celery chromosome, Chinese cabbage chromosome, cherry chromosome, coriander leaf chromosome, citrus chromosome, clementine chromosome, coffee chromosome, corn chromosome, cotton chromosome, cucumber chromosome, Douglas fir chromosome, eggplant chromosome, endive chromosome, chrysanthemum chromosome, eucalyptus chromosome, fennel chromosome, fig chromosome, forest tree chromosome, gourd chromosome, grape chromosome, grapefruit chromosome, honeydew chromosome, jicama chromosome, kiwi chromosome, lettuce chromosome, chive chromosome, lemon chromosome, lime chromosome, taeda Pine chromosome, mango chromosome, maple chromosome, melon chromosome, mushroom chromosome, nectarine chromosome, nut chromosome, oat chromosome, okra chromosome, onion chromosome, orange chromosome, plant chromosome, papaya chromosome, parsley chromosome, pea chromosome, peach chromosome, peanut chromosome, pear chromosome, pepper chromosome, persimmon chromosome, pine chromosome, pineapple chromosome, plantain chromosome, plum chromosome, pomegranate chromosome, poplar chromosome, potato chromosome, pumpkin chromosome, quince chromosome, radiata pine chromosome, radiata The group is selected from the following: kio chromosome, radish chromosome, rapeseed chromosome, raspberry chromosome, rice chromosome, rye chromosome, sorghum chromosome, southern pine chromosome, soybean chromosome, spinach chromosome, melon chromosome, strawberry chromosome, sugar beet chromosome, sugarcane chromosome, sunflower chromosome, sweet corn chromosome, sweet potato chromosome, sweet leaf chromosome, tangerine chromosome, brown chromosome, tobacco chromosome, tomato chromosome, grass chromosome, climbing plant chromosome, watermelon chromosome, wheat chromosome, yam chromosome, and zucchini chromosome.

[0128] In one aspect, the cells provided herein are modified cells. In another aspect, the plants provided herein are modified plants. In yet another aspect, the plant cells provided herein are modified plant cells. In yet another aspect, the seeds provided herein are modified seeds. In yet another aspect, the chromosomes provided herein are modified chromosomes.

[0129] According to one embodiment, a modified plant, plant cell, cell, seed, or chromosome provided herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten deletions generated by targeted editing techniques. According to one embodiment, a modified plant, plant cell, cell, seed, or chromosome provided herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten insertions generated by targeted editing techniques. According to one embodiment, a modified plant, plant cell, cell, seed, or chromosome provided herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten inversions generated by targeted editing techniques. According to one embodiment, the modified plants, plant cells, cells, seeds, or chromosomes provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten deletions, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten insertions, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten inversions, or any combination thereof, generated by targeted editing techniques. In yet another embodiment, the modified plants, plant cells, cells, seeds, or chromosomes provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten dominant-negative alleles generated by targeted editing techniques.In yet another embodiment, the modified plants, plant cells, cells, seeds, or chromosomes provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten dominant-positive alleles generated by targeted editing technology. In yet another embodiment, the modified plants, plant cells, cells, seeds, or chromosomes provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten dominant-negative alleles generated by targeted editing technology, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten dominant-positive alleles generated by targeted editing technology, or any combination thereof.

[0130] According to another aspect of this application, modified plants(s), plant cells(s), seeds(s), chromosomes(s), and plant parts(s) are provided, which include a genome editing event, including insertion, deletion, substitution, or inversion of a targeted locus, in the genome of at least one plant cell.

[0131] In one aspect, the Disclosure provides modified plant cells produced by any one of the methods provided herein. In another aspect, the Disclosure provides modified chromosomes produced by any one of the methods provided herein. In yet another aspect, the Disclosure provides modified cells containing modified chromosomes provided herein. In yet another aspect, the Disclosure provides modified plants or modified plant tissues regenerated from modified cells provided herein. In yet another aspect, the Disclosure provides products containing modified chromosomes provided herein. In one aspect, the Disclosure provides products containing modified cells provided herein. As used herein, “product” means any article or substance intended for human use, human consumption, animal use, or animal consumption, including any components, parts, or appendages containing modified cells or modified chromosomes provided herein.

[0132] The methods and compositions provided herein can edit any locus within a genome. Chromosomes edited by using the methods and compositions provided herein are also provided herein. In one embodiment, the genome provided herein is a nuclear genome, a mitochondrial genome, or a plastid genome. In another embodiment, the plastid genome provided herein includes a chloroplast genome. In one embodiment, the method provided herein generates a double-strand break on a chromosome. In one embodiment, the chromosome provided herein is a nuclear chromosome, a mitochondrial chromosome, or a chloroplast chromosome. In another embodiment, the chromosome provided herein is a superchromosome or artificial chromosome. A superchromosome, or B chromosome, is an extra chromosome found in addition to the normal diploid complement of chromosomes within a cell. Superchromosomes are deficient and not required for the normal development of a cell or organism. In one embodiment, the superchromosome provided herein is a maize superchromosome or a rye superchromosome.

[0133] Methods of targeted editing disclosed herein may include transient transfection or stable transformation of cells of interest (e.g., plant cells). One aspect of this application provides a method for producing transgenic or genome-edited cells by transforming cells, tissues, or explants with a recombinant DNA molecule or construct containing a transgenic DNA sequence or transgene operably linked to a promoter. Another aspect of this application provides a method for producing transgenic or genome-edited plants or plant cells by transforming plant cells, tissues, or explants with a recombinant DNA molecule or construct containing a transgenic DNA sequence or transgene operably linked to a promoter expressible in plants. As used herein, “transgene” means a polynucleotide introduced into the genome by any method known in the art.

[0134] Numerous methods for transforming chromosomes or plastids within plant cells using recombinant DNA molecules or constructs are known in the art, and these can be used according to the method of this application to produce transgenic plant cells and plants. Any suitable method or technique for transforming plant cells known in the art can be used according to the method of the present invention. Effective methods for transforming plants include bacterial-mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation, and microparticle gun-mediated transformation. Various methods for regenerating or developing transgenic plants are known in the art, such as transforming explants with transformation vectors via bacterial-mediated transformation or microparticle guns, and then culturing these explants. Other plant transformation methods, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, and PEG-mediated transformation, are also known in the art. Transgenic plants produced by these transformation methods may be chimeric or non-chimeric with respect to the transformation event, depending on the method and explant used.

[0135] Methods for transforming plant cells are well known to those skilled in the art. For example, specific descriptions for transforming plant cells by a particulate gun using recombinant DNA-coated particles can be found in U.S. Patents 5,550,318, 5,538,880, 6,160,208, 6,399,861 and 6,153,812, and for Agrobacterium-mediated transformation, U.S. Patents 5,159,135, 5,824,877, 5,591,616, 6,384,301, 5,750,871, 5,463,174 and 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009), Blackwell Publishing. Any suitable method known to those skilled in the art can be used to transform plant cells with any of the nucleic acid molecules provided herein.

[0136] Examples of recipient cells or explant targets for transformation include, but are not limited to, seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endomass cells, root cells, shoot cells, stem cells, pod cells, flower cells, inflorescence cells, stem cells, root cells, style cells, stigma cells, bed cells, petal cells, sepal cells, pollen cells, anther cells, peduncle cells, ovary cells, ovule cells, pericarp cells, phloem cells, bud cells, or vascular tissue cells. In another embodiment, the disclosure provides plant chloroplasts. In a further embodiment, the disclosure provides epidermal cells, stomatal cells, trichome cells, root hair cells, storage root cells, or tuber cells. In another embodiment, the disclosure provides protoplasts. In another embodiment, the disclosure provides plant callus cells. Any cell from which a fertile plant can be regenerated is considered useful as a recipient cell in the practice of the disclosure. Callus can be initiated from a variety of tissue sources, including but not limited to immature embryos or embryonic portions, seedling apical meristems, and microspores. These cells, which can proliferate as callus, can serve as recipient cells for transformation. Actual transformation methods and materials for producing transgenic plants of this disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Transformed explants, cells, or tissues may be subjected to additional culture steps, such as callus induction, selection, and regeneration, as known in the art. Transformed cells, tissues, or explants containing recombinant DNA insertions can be grown, developed, or regenerated into transgenic plants in culture, plugs, or soil according to methods known in the art. In one embodiment, the disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of plants. In another embodiment, the disclosure also provides plant cells that are reproductive material and mediate the natural reproduction of plants. In yet another embodiment, the disclosure provides plant cells that are unable to sustain themselves through photosynthesis.In another embodiment, the disclosure provides somatic plant cells. Unlike germ cells, somatic cells do not mediate reproduction in plants. In one embodiment, the disclosure provides non-germinal plant cells.

[0137] Modified plants may be further crossbred with themselves or other plants to produce modified seeds and offspring. Modified plants can also be prepared by crossing a first plant containing a recombinant DNA sequence insertion with a second plant lacking the insertion. For example, a recombinant DNA sequence can be introduced into a first plant line suitable for transformation, and then this can be crossbred with a second plant line to transfer the recombinant DNA sequence into the second plant line. Modified plants can also be prepared by crossing modified plants with unmodified plants. The offspring of these crosses may be further backcrossed multiple times, such as 6-8 generations or backcrosses, to produce offspring plants that have substantially the same genotype as the original parent line but are suitable for introducing recombinant DNA constructs or modified sequences.

[0138] The modified plants, cells, or explants provided herein may be of elite varieties or elite lines. Elite varieties or lines refer to any variety resulting from breeding and selection for superior agricultural performance. The modified plants, cells, or explants provided herein may be hybrid plants, cells, or explants. As used herein, a “hybrid” is produced by crossing two plants of different varieties, lines, or species such that the offspring contain genetic material from each parent. Those skilled in the art will recognize that higher-order hybrids can also be produced. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C×D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E×F hybrid. The first and second hybrids may be further crossed to produce a higher-order hybrid (C×D)×(E×F) containing genetic information from all four parent varieties. The modified plants provided herein are fertile. The modified plants provided herein are male or female sterile modified plants that cannot reproduce without human intervention. In one embodiment, the modified plants provided herein reproduce asexually or vegetatively. In yet another embodiment, the modified plants provided herein reproduce sexually.

[0139] The recombinant DNA molecules or constructs of this application include, or may include, DNA transformation vectors for use in transforming target plant cells, tissues, or explants. Such transformation vectors of this application may generally include, at least one select marker gene, at least one expression cassette, and / or one or more site-specific nucleases encoded in a transcribed DNA sequence, and optionally, one or more sgRNAs or crRNAs, in addition to sequences or elements necessary or beneficial for effective transformation. In the case of Agrobacterium-mediated transformation, the transformation vector may include an engineered transfer DNA (or T-DNA) segment or region having two boundary sequences, a left boundary (LB) and a right boundary (RB), that flank to at least a transcribed DNA sequence or transgene, such that insertion of the T-DNA into the plant genome results in a transformation event of the transcribed DNA sequence, transgene, or expression cassette. In other words, the transgene, transgenic DNA sequence, expression cassette encoding the transgene or site-specific nuclease(s), and / or sgRNA(s) or crRNA(s) are located between the left and right boundaries of the T-DNA, along with additional transgene(s) or expression cassette(s), such as plant-selection marker transgenes and / or other genes(s) of agricultural interest that may confer traits or phenotypes of agricultural interest to the plant. In an alternative embodiment, the transgenic DNA sequence, transgene or expression cassette encoding at least one site-specific nuclease, any required sgRNA or crRNA, and plant-selection marker transgene(s) (or other genes of agricultural interest) may reside in separate T-DNA segments of the same or different recombinant DNA molecule(s), for example, for simultaneous transformation. The transformation vector or construct may further include prokaryotic maintenance elements, which, in the case of Agrobacterium-mediated transformation, may be located within the vector skeleton outside the T-DNA region(s).

[0140] If a plant selection marker transgene confers tolerance or resistance to a selective agent, the plant selection marker transgene in the transformation vector or construct of this application can be used to support the selection of transformed cells or tissues in the presence of a selective agent such as an antibiotic or herbicide. Thus, the selective agent can bias or prioritize the survival, development, growth, proliferation, etc., of transformed cells expressing the plant selection marker gene, for example, increasing the proportion of transformed cells or tissues in R0 plants. Commonly used plant selection marker genes include those that confer tolerance or resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aadA) and gentamicin (aac3 and aacC4), or those that confer tolerance or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or Cp4-EPSPS). Plant screening marker genes that enable visual screening of transformants, such as luciferase or green fluorescent protein (GFP), or genes expressing beta-glucuronidase or the uidA gene (GUS), for which various chromogenic substrates are known, may be used. In one embodiment, the vector or polynucleotide provided herein comprises at least one marker gene selected from the group consisting of nptII, aph IV, aadA, aac3, aacC4, bar, pat, DMO, EPSPS, aroA, GFP, and GUS.

[0141] According to certain embodiments of this application, methods for transforming plant cells, tissues, or explants using recombinant DNA molecules or constructs may further include site-directed or targeted integration using site-specific nucleases. These methods allow for the insertion or integration of a portion of a recombinant DNA donor molecule (i.e., an insertion sequence) at a desired site or locus within the genome. The insertion sequence of the donor template may include a transgene or construct, such as a designed element or a tissue-specific promoter. The donor molecule may also have one or two homologous arms that flank the insertion sequence to enhance targeted insertion events via homologous recombination and / or homologous directing repair. Thus, the recombinant DNA molecules of this application may further include donor templates for site-directed or targeted integration into the genome of transgenes or constructs, such as transgenes or transcriptable DNA sequences encoding a designed element or a tissue-specific promoter.

[0142] As used herein, a “part” of a nucleic acid sequence or molecule refers to any number of nucleotides shorter than the full length of the nucleic acid sequence. For example, a part of a 100-nucleotide nucleic acid sequence could be any number of nucleotides from 1 to 99. Alternatively, a “part” of a nucleic acid sequence refers to any range from 0.01% to 99.99% of the full length of a given nucleic acid sequence.

[0143] This specification provides a method for generating a dominant allele of a gene region using targeted editing techniques. This specification also provides cells produced by such a method and the composition used in such a method. This specification further provides modified plants regenerated from cells subjected to the method provided herein. In one embodiment, the dominant-negative allele provided herein can repress the transcription of a heterozygous locus or gene. In another embodiment, the dominant-negative allele provided herein can repress the transcription of a homozygous locus or gene.

[0144] Dominant-negative alleles of a gene region can reduce or eliminate the function of heterozygous gene region products. Dominant-negative alleles can be generated by editing the allele of a gene region such that the orientation of at least a portion of the polynucleotide encoding the gene region is reversed (for example, a portion of the gene is reversed from 3' to 5' orientation, while the remainder remains oriented from 5' to 3'). Expression of the edited allele of a gene region will include an antisense RNA segment complementary to the sense RNA expressed by the unedited gene region. Without being bound by any scientific theory, processing the complementary segment between the sense and antisense portions of the gene region RNA by a cell-specific RNA silencing mechanism may reduce the expression of both edited and unedited gene region alleles in a dominant-negative manner. In one embodiment, the antisense RNA transcript provided herein can repress a complementary sense RNA transcript. In another embodiment, the antisense RNA transcript provided herein represses a complementary sense RNA transcript.

[0145] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising using a targeted editing technique to invert a portion of the gene to produce an antisense RNA transcript that can induce repression of the unmodified allele of the gene. In one embodiment, expression of the unmodified allele is reduced compared to a control cell without the antisense RNA transcript. In another embodiment, the targeted editing technique provided herein comprises the use of at least one site-specific nuclease. In one embodiment, the antisense RNA transcript provided herein is a partial antisense RNA transcript. In another embodiment, the antisense RNA transcript provided herein is a complete antisense RNA transcript. A partial antisense RNA transcript may be generated by inverting only one region of the gene, rather than inverting the entire gene. For example, if an mRNA transcript is encoded by three exons, it may be possible to generate a partial antisense RNA transcript by inverting only the second exon. As can be understood, inverting any number of nucleotides in a gene region that is shorter than the total length of the gene region can produce a partial antisense RNA transcript. For example, if a gene region contains 500 nucleotides, inverting a 200-nucleotide region will produce a partial antisense RNA transcript. Inverting all 500 nucleotides will produce a complete antisense RNA transcript. In some embodiments, the antisense RNA transcripts provided herein can suppress the expression of complementary nucleic acid sequences. In some embodiments, the antisense RNA transcripts provided herein can suppress the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes. In some embodiments, the antisense RNA transcripts provided herein can suppress the expression of a first gene region. In some embodiments, the antisense RNA transcripts provided herein can suppress the expression of a protein encoded by a complementary nucleic acid sequence.As will be apparent to those skilled in the art, 100% complementarity between the antisense RNA transcript and the second nucleic acid is not required to induce repressed expression of the second nucleic acid. For example, an antisense RNA transcript containing at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% complementarity with respect to the second nucleic acid sequence may be able to repress the expression of the second nucleic acid sequence.

[0146] In one embodiment, an antisense RNA transcript transcribed by a dominant-negative allele provided herein can downregulate its own expression. In another embodiment, an antisense RNA transcript transcribed by a dominant-negative allele provided herein can downregulate the expression of an unmodified allele at the same locus. In one embodiment, the expression of the unmodified allele is reduced compared to control cells without the antisense RNA transcript.

[0147] In one embodiment, the Disclosure provides a method for generating a dominant-negative allele of a gene in one or more cells, comprising: a) inducing a first double-strand break and a second double-strand break that flank in the targeting region of the gene; b) identifying one or more cells containing an inversion of the targeting region of the gene, wherein the inversion results in the production of an antisense RNA transcript from the targeting region of the gene; and c) selecting one or more cells containing the inversion of the targeting region of the gene.

[0148] In another embodiment, the Disclosure provides a method for reducing protein expression in cells, comprising: a) inducing a first double-strand break and a second double-strand break that flank a chromosomal targeting region; and b) identifying one or more cells containing an inversion in the chromosomal targeting region, wherein protein expression is reduced compared to control cells that do not contain an inversion in the targeting region.

[0149] In a further embodiment, the Disclosure provides a method for generating an inversion in a targeted region of a gene, comprising: a) providing one or more cells with at least one RNA guide nuclease, or one or more vectors encoding an RNA guide nuclease, such that the at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides of a first target site and a second target site flanking in the targeted region of a gene, the first target site and the second target site being linked, and the at least one RNA guide nuclease producing a double-strand break at the first target site and the second target site of the gene; b) identifying one or more cells containing an inversion in the targeted region of a gene, such that the inversion results in the production of an antisense RNA transcript from the targeted region; and c) selecting one or more cells containing an inversion in the targeted region of a gene.

[0150] In one embodiment, the method or composition provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 site-specific nucleases. In another embodiment, the method or composition provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sgRNAs. In yet another embodiment, the method or composition provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 tgOligos. In yet another embodiment, the method or composition provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 donor molecules. In another embodiment, the methods or compositions provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten donor sequences.

[0151] In another embodiment, a method or composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten vectors encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. In another embodiment, a method or composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten vectors encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sgRNAs. In another embodiment, a method or composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten vectors encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten tgOligo molecules. In another embodiment, a method or composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten vectors encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten donor molecules.In another embodiment, the methods or compositions provided herein include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten vectors encoding at least one, at least two, at least three, at least four, at least six, at least seven, at least eight, at least nine, or at least ten donor sequences.

[0152] In one embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five site-specific nucleases. In one embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five RNA guide nucleases. In another embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five sgRNAs. In one embodiment, the method or composition provided herein comprises one or more vectors containing a first sgRNA and a second sgRNA. In a further embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five donor molecules.

[0153] In one embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five site-specific nucleases and at least one, at least two, at least three, at least four, or at least five sgRNAs. In another embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five RNA guide nucleases and at least one, at least two, at least three, at least four, or at least five sgRNAs. In one embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five sgRNAs and at least one, at least two, at least three, at least four, or at least five donor molecules.

[0154] In another embodiment, the vector provided herein encodes at least one RNA guide nuclease, a first sgRNA, and a second sgRNA. In a further embodiment, at least one RNA guide nuclease, a first sgRNA, and a second sgRNA are encoded by two or more or three or more vectors. In another embodiment, the vector provided herein encodes at least one RNA guide nuclease, an sgRNA, and a donor molecule. In a further embodiment, at least one RNA guide nuclease, an sgRNA, and a donor molecule are encoded by two or more or three or more vectors.

[0155] In another embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five site-specific nucleases and at least one, at least two, at least three, at least four, or at least five donor molecules. In one embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five RNA guide nucleases and at least one, at least two, at least three, at least four, or at least five donor molecules. In another embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five site-specific nucleases, at least one, at least two, at least three, at least four, or at least five sgRNAs and at least one, at least two, at least three, at least four, or at least five donor molecules. In another embodiment, the vector provided herein encodes at least one, at least two, at least three, at least four, or at least five RNA guide nucleases, at least one, at least two, at least three, at least four, or at least five sgRNAs, and at least one, at least two, at least three, at least four, or at least five donor molecules. In another embodiment, the vector provided herein encodes at least one site-specific nuclease, at least one donor molecule, and at least one sgRNA. In another embodiment, the vector provided herein encodes at least one RNA guide nuclease, at least one donor molecule, and at least one sgRNA.

[0156] In one embodiment, one or more site-specific nucleases, one or more sgRNAs, and one or more donor molecules provided herein are encoded by one vector. In one embodiment, one or more site-specific nucleases, one or more sgRNAs, and one or more donor molecules provided herein are encoded by two or more or three or more vectors. In yet another embodiment, one or more sgRNAs and one or more donor molecules provided herein are encoded by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more vectors. In one embodiment, at least one RNA guide nuclease, a first sgRNA, and a second sgRNA are encoded by one vector. In another embodiment, at least one RNA guide nuclease, a first sgRNA, and a second sgRNA are encoded by two or more or three or more vectors. In one embodiment, at least one site-specific nuclease, a first sgRNA, and a second sgRNA are encoded by one vector. In another embodiment, at least one site-specific nuclease, a first sgRNA, and a second sgRNA are encoded by two or more or three or more vectors. In one embodiment, at least one RNA guide nuclease, at least one sgRNA, and at least one donor molecule are encoded by one vector. In one embodiment, at least one RNA guide nuclease, at least one sgRNA, and at least one donor molecule are encoded by two or more or three or more vectors. In one embodiment, at least one site-specific nuclease, at least one sgRNA, and at least one donor molecule are encoded by one vector. In one embodiment, at least one site-specific nuclease, at least one sgRNA, and at least one donor molecule are encoded by two or more or three or more vectors.

[0157] In one embodiment, one or more Cas9 nucleases, one or more sgRNAs, and one or more donor molecules provided herein are encoded by one vector. In one embodiment, one or more Cas9 nucleases, one or more sgRNAs, and one or more donor molecules provided herein are encoded by two or more or three or more vectors. In one embodiment, at least one Cas9 nuclease, a first sgRNA, and a second sgRNA are encoded by one vector. In another embodiment, at least one Cas9 nuclease, a first sgRNA, and a second sgRNA are encoded by two or more or three or more vectors. In one embodiment, at least one Cas9 nuclease, at least one sgRNA, and at least one donor molecule are encoded by one vector. In one embodiment, at least one Cas9 nuclease, at least one sgRNA, and at least one donor molecule are encoded by two or more or three or more vectors.

[0158] In yet another embodiment, any vector described herein further encodes at least one, at least two, at least three, at least four, or at least five marker genes. In one embodiment, the marker genes provided herein are selected from the group consisting of nptII, aph IV, aadA, aac3, aacC4, bar, pat, DMO, EPSPS, aroA, GFP, and GUS.

[0159] Targeted editing techniques allow for the transformation of genomic loci into loci capable of generating RNAi-inducible hairpins when the edited loci are transcribed into RNA. In cells that are heterozygous at the target locus (e.g., two polymorphic alleles exist), two or more nucleases are used to generate two double-strand breaks in the first allele (e.g., a first and a second double-strand break) and one double-strand break in the second allele (e.g., a third double-strand break). When the nucleases cleave the alleles, portions of the first allele that are flanked into the first and second double-strand breaks are released from the genomic DNA. In one result, the released portion of the first allele is inverted and incorporated into the third double-strand break in the second allele, thereby creating an edited locus capable of generating RNAi-inducible hairpins when the edited locus is transcribed.

[0160] This disclosure provides a method comprising: a) using targeted editing technology to generate a first double-strand break and a second double-strand break in a first allele of a gene in a cell; using targeted editing technology to generate a third double-strand break in a second allele of a gene in a cell; and c) identifying a cell that contains an insertion of a region of the first allele in the opposite direction at the site of the third double-strand break in the second allele, thereby generating a modified second allele. In one embodiment, the modified second allele is a dominant-negative allele. In another embodiment, the modified second allele is a dominant-positive allele. In one embodiment, the first double-strand break and the second double-strand break are located in the same nucleotide sequence or at the same nucleotide position in the first and second alleles. In one embodiment, the first double-strand break and the second double-strand break are located in the same nucleotide sequence in the first and second alleles. In one embodiment, the first and second double-strand breaks are located at the same nucleotide positions in the first and second alleles. In one embodiment, the nucleotide sequence of the first allele is not identical to the nucleotide sequence of the second allele (for example, cells are heterozygous for their loci). In one embodiment, the nucleotide sequence of the third double-strand break site in the second allele is absent in the first allele. In one embodiment, the modified second allele provided herein transcribes RNA capable of forming a hairpin loop secondary structure. In one embodiment, the region of the first allele may contain any number of nucleotides up to the full length of the first allele.In one embodiment, the region of the first allele contains at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 50, and a small number of It contains at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In another embodiment, the region of the first allele contains 18-5000, 18-4000, 18-3000, 18-2000, 18-1000, 18-500, 18-400, 18-300, 18-200, 18-100, 18-50, 18-30, 50-500, 50-1000, 100-500, 100-1000, or 500-5000 nucleotides.

[0161] In one embodiment, the disclosure provides a modified cell comprising at least one dominant-negative allele of at least one gene generated by a targeted editing technique, wherein, upon transcription of the at least one dominant-negative allele, the allele generates an RNA transcript capable of forming a hairpin loop secondary structure.

[0162] This disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising using targeted editing techniques to insert an inverted copy of the gene or a portion thereof adjacent to the native copy of the gene to generate an inverted repeat sequence capable of producing an antisense RNA transcript of the gene or a portion thereof. In one embodiment, the inverted repeat sequence can form a hairpin loop secondary structure. In another embodiment, the dominant-negative allele generates at least one RNA transcript capable of forming a hairpin loop secondary structure. In one embodiment, the inverted copy of the gene and the native copy of the gene are separated by a spacer sequence. In one embodiment, the spacer sequence contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 250, at least 500, or at least 1000 nucleotides. In yet another embodiment, the dominant-negative allele is operably ligated to the promoter of the native copy of the gene.

[0163] In another embodiment, the disclosure provides a modified cell comprising a dominant-negative allele of at least one gene and an inverted copy of the gene adjacent to the native copy of the gene at the endogenous locus of the gene.

[0164] Dominant-negative alleles can also be generated by editing the genome to delete a region between the first gene region and a second neighboring gene region of DNA, so that the first and second gene regions are oriented in opposite directions on the chromosome (for example, the first gene region is oriented 5' to 3' and the second gene region is oriented 3' to 5' on the same DNA strand of the chromosome). While not bound by any particular scientific theory, such a deletion allows the promoter of the second gene region to express the antisense RNA transcript of the first gene region, while the native promoter of the first gene region expresses the sense RNA transcript. Since the sense and antisense RNA transcripts are complementary, processing them by cell-specific RNA silencing mechanisms can reduce the expression of both the edited and unedited first gene region alleles in a dominant-negative manner. While not bound by any particular scientific theory, it is also thought that antisense RNA molecules transcribed from endogenous genes or loci mutations or edited alleles may affect gene expression levels through a variety of mechanisms, including nonsense-dependent decay, nonstop decay, no-go decay, DNA or histone methylation or other epigenetic changes, inhibition or reduction of transcription and / or translation efficiency, ribosome interference, interference with mRNA processing or splicing, and / or ubiquitin-mediated proteasome degradation.See, for example, Nickless, A. et al., “Control of gene expression through the nonsense-mediated RNA decay pathway”, Cell Biosci 7:26 (2017), Karamyshev, A. et al., “Lost in Translation: Ribosome-Associated mRNA and Protein Quality Controls”, Frontiers in Genetics 9:431 (2018), Inada, T., “Quality controls induced by aberrant translation”, Nucleic Acids Res 48:3 (2020), and Szadeczky-Kardoss, I. et al., “The nonstop decay and the RNA silencing systems operate cooperatively in plants”, Nucleic Acids Res 46:9 (2018). The contents and disclosures of these works are incorporated herein by reference. These different mechanisms may act instead of, or in addition to, RNA interference (RNAi), transcriptional gene silencing (PGS), and / or post-transcriptional gene silencing (PTGS) mechanisms. See, for example, Wilson, RC et al., “Molecular Mechanisms of RNA Interference”, Annu Rev Biophysics 42:217-39 (2013), and Guo, Q. et al., “RNA Silencing in Plants: Mechanism, Technologies and Applications in Horticulture Crops”, Current Genomics 17:476-489 (2016). The contents and disclosures of these works are incorporated herein by reference. Some of the above mechanisms may reduce the expression of the edited allele itself, while others may reduce the expression of other copies of an endogenous locus or gene or allele.Such dominant or semi-dominant effects on a gene(s) may act through non-standard repressive mechanisms that do not involve the formation of RNAi and / or targeted small RNAs at significant or detectable levels.

[0165] In one embodiment, the disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising deleting a portion of a chromosome between a first gene region and a second gene region using a targeted editing technique, wherein an antisense RNA transcript of the first gene region is generated after the deletion of the chromosomal portion. In another embodiment, the targeted editing technique provided herein comprises the use of at least one site-specific nuclease. In one embodiment, the antisense RNA transcript provided herein is a partial antisense RNA transcript. In one embodiment, the partial antisense RNA transcript is shorter than the corresponding sense RNA transcript. In one aspect, a partial antisense RNA transcript is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, or at least 2500 nucleotides shorter than the corresponding sense RNA transcript. In another embodiment, a partial antisense RNA transcript is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% shorter than the corresponding sense RNA transcript. In another embodiment, the antisense RNA transcript provided herein is a complete antisense RNA transcript. In one embodiment, the complete antisense RNA transcript is the same length as the corresponding sense RNA transcript. In one embodiment, the antisense RNA transcript provided herein represses the expression of a first gene region.In one embodiment, the antisense RNA transcript provided herein can suppress the expression of a first gene region.

[0166] In another embodiment, the Disclosure provides a method comprising: a) identifying a chromosomal region comprising a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region and the first and second promoters are positioned in opposite directions; b) inducing a first double-strand break and a second double-strand break that flank a targeting region; c) identifying one or more cells comprising a deletion of a chromosomal targeting region; and d) selecting one or more cells comprising a deletion of a chromosomal targeting region.

[0167] As used herein, “intervening region” or “intervening sequence” refers to a polynucleotide sequence between a physically linked first polynucleotide sequence and a second polynucleotide sequence. In one embodiment, the intervening region or intervening sequence is located between a first gene and a second gene. In one embodiment, the intervening region or intervening sequence is located between a first gene region and a second gene region. In one embodiment, the intervening region or intervening sequence is located between a first coding region and a second coding region. In another embodiment, the intervening region or intervening sequence is located between a first target site and a second target site. In one embodiment, the intervening region or intervening sequence is located between a first target gene and a second target gene. In one embodiment, all or part of the intervening region or intervening sequence is inverted by targeted editing techniques. In another embodiment, all or part of the intervening region or intervening sequence is deleted by targeted editing techniques. In one embodiment, the intervening region or intervening sequence contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, or at least 50,000 nucleotides. In one embodiment, the intervening region or intervening sequence contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes. In one embodiment, the intervening region or intervening sequence is located on a chromosome. In one embodiment, the intervening region or intervening sequence is located on a vector. In one embodiment, the intervening region or intervening sequence contains a DNA sequence. In one embodiment, the intervening region or intervening sequence contains an RNA sequence.In one embodiment, the intervening region or intervening sequence includes an endogenous nucleic acid sequence. In another embodiment, the intervening region or intervening sequence includes a transgenic nucleic acid sequence. In one embodiment, the intervening region or intervening sequence includes an endogenous nucleic acid sequence and a transgenic nucleic acid sequence.

[0168] In one embodiment, the first gene region is selected from the group consisting of the GA20 oxidase gene region, the GA3 oxidase gene region, the brachytic2 gene region, and the Y1 gene region. In another embodiment, the first gene region is the GA20 oxidase gene region or the GA3 oxidase gene region. In yet another embodiment, the first gene region is the GA20 oxidase gene region. In one embodiment, the first gene region is the GA3 oxidase gene region. In yet another embodiment, the first gene region is the brachytic2 gene region. In yet another embodiment, the first gene region is the Y1 gene region.

[0169] GA oxidases in cereal plants consist of a family of related GA oxidase genes. For example, maize has a family of at least nine GA20 oxidase genes, including GA20 oxidase_1, GA20 oxidase_2, GA20 oxidase_3, GA20 oxidase_4, GA20 oxidase_5, GA20 oxidase_6, GA20 oxidase_7, GA20 oxidase_8, and GA20 oxidase_9. The DNA and protein sequences of GA20 oxidase_3 and GA20 oxidase_5, according to their respective sequence numbers, are provided in Table 1. [Table 1]

[0170] The wild-type genomic DNA sequence for the GA20 oxidase_3 locus from the reference genome is provided as SEQ ID NO: 27, and the wild-type genomic DNA sequence for the GA20 oxidase_5 locus from the reference genome is provided as SEQ ID NO: 31.

[0171] For the maize GA20 oxidase_3 gene (also known as Zm.GA20ox3), Sequence ID 27 provides the 3000 nucleotides upstream (5′) of the 5′-UTR of GA20 oxidase_3, with nucleotides 3001-3096 corresponding to the 5′-UTR, nucleotides 3097-3665 corresponding to the first exon, nucleotides 3666-3775 corresponding to the first intron, nucleotides 3776-4097 corresponding to the second exon, nucleotides 4098-5314 corresponding to the second intron, nucleotides 5315-5584 corresponding to the third exon, and nucleotides 5585-5800 corresponding to the 3′-UTR. Sequence ID 27 also provides the 3000 nucleotides downstream (3′) of the 3′-UTR (nucleotides 5801-8800).

[0172] For the maize GA20 oxidase_5 gene (also known as Zm.GA20ox5), Sequence ID 31 provides the first 3000 nucleotides upstream of the GA20 oxidase_5 start codon (nucleotides 1-3000), with nucleotides 3001-3791 corresponding to the first exon, nucleotides 3792-3906 corresponding to the first intron, nucleotides 3907-4475 corresponding to the second exon, nucleotides 4476-5197 corresponding to the second intron, nucleotides 5198-5473 corresponding to the third exon, and nucleotides 5474-5859 corresponding to the 3′-UTR. Sequence ID 31 also provides the last 3000 nucleotides downstream of the 3′-UTR (3′) (nucleotides 5860-8859).

[0173] In the maize genome, the Zm.GA20ox5 gene is located adjacent to the Zm.SAMT gene. These two genes are separated by an intergenetic region of approximately 550 bp, with the Zm.SAMT gene located downstream of the Zm.GA20ox5 gene and oriented in the opposite direction. Reference genome sequences of the region encompassing the Zm.GA20ox5 and Zm.SAMT genes are provided in SEQ ID NOs. 35 and 36. SEQ ID NOs. 35 represents the sequence of the sense strand of the Zm.GA20ox5 gene, encompassing both the Zm.GA20ox5 and Zm.SAMT genes (see "GA20ox5_SAMT Genome Sequence" in Table 2). SEQ ID NOs. 35 partially overlaps with SEQ ID NOs. 31 and has a shorter Zm.GA20ox5 upstream sequence and a longer Zm.GA20ox5 downstream sequence compared to SEQ ID NOs. 31. Sequence ID 36 represents the sequence of the sense strand of the Zm.SAMT gene (i.e., the antisense strand of the Zm.GA20ox5 gene), which encompasses both the Zm.GA20ox5 and Zm.SAMT genes (see "SAMT_GA20ox5 Genome Sequence" in Table 2). Table 2 below annotates elements or regions of the reference genome Zm.GA20ox5 / Zm.SAMT sequence by referring to the nucleotide coordinates of these elements or regions in Sequence ID 35 or 36.

[0174] Transgenic repression of the GA20 oxidase gene(s) and / or targeting of one or more subsets of GA20 oxidase genes (e.g., artificial microRNA-mediated repression of both the GA20 oxidase_3 and GA20 oxidase_5 genes) has previously been shown to be effective in achieving a low-stance, semi-dwarf phenotype with increased lodging resistance but no reproductive dysplasia in the panicles. See PCT application PCT / US2017 / 047405 and US application 15 / 679,699, both filed on August 17, 2017, and published as WO / 2018 / 035354 and US20180051295, respectively. Furthermore, genome editing knockout of GA20 oxidase_3, GA20 oxidase_5, or both genes can also affect GA hormone levels, resulting in reduced plant height and increased lodging resistance. See PCT applications PCT / US2019 / 018128, PCT / US2019 / 018131, and PCT / US2019 / 018133, all filed on February 15, 2019.

[0175] In one embodiment, the first gene region includes a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity or complementarity with a sequence selected from the group consisting of sequence numbers (inserting GA20 cDNA sequences).

[0176] In another embodiment, the first gene region comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity or complementarity with a sequence selected from the group consisting of sequence numbers (insertion of BR2 cDNA sequences).

[0177] In one embodiment, the first gene region includes a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity or complementarity with a sequence selected from the group consisting of sequence numbers (insertion of GA3 cDNA sequence).

[0178] In one embodiment, the first gene region includes a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity or complementarity with a sequence selected from the group consisting of sequence numbers (insertion of Y1 cDNA sequence).

[0179] In one embodiment, the deletion provided herein includes all or part of the second gene region. In another embodiment, the deletion provided herein includes all of the second gene region. In yet another embodiment, the deletion provided herein includes part of the second gene region.

[0180] In yet another embodiment, the Disclosure provides a method for reducing gene expression in cells, comprising: a) identifying a chromosomal region comprising a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region, and the first and second promoters are positioned in opposite directions; b) inducing a first double-strand break and a second double-strand break that flanks a targeting region using a targeted editing technique, wherein the targeting region comprises a second coding region and an intervening region; and c) identifying one or more cells containing a deletion of a chromosomal targeting region, wherein the second promoter generates at least one antisense RNA of the first coding region, and the expression of the first coding region is reduced compared to control cells that do not contain the deletion of the targeting region. In one embodiment, the deletion leads to the reverse transcription of a portion of the first coding region.

[0181] In a further embodiment, the Disclosure provides a) identification of a chromosomal region comprising a first gene region comprising a first promoter and a first coding region and a second gene region comprising a second promoter and a second coding region, wherein the first and second coding regions are separated by an intervening region and the first and second promoters are positioned in opposite directions, and b) providing one or more cells with at least one RNA guide nuclease, or one or more vectors encoding at least one RNA guide nuclease, The present invention provides an RNA guide nuclease that can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides of a first target site and a second target site that flank a targeting region of a chromosome, wherein the targeting region comprises a second coding region and an intervening region, and the RNA guide nuclease produces a double-strand break at the first target site and the second target site of the chromosome; and a method is provided comprising a) identifying one or more cells containing a deletion of the targeting region; and c) selecting one or more cells containing a deletion of the targeting region.

[0182] In one embodiment, the disclosure provides a modified plant or part thereof comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the disclosure provides a modified plant cell comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In another embodiment, the disclosure provides a modified plant or modified plant tissue comprising a modified plant cell comprising a modified plant cell comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof.

[0183] In the art, it is known that transposons, or transposition factors, are DNA sequences that can change position within the genome. Transposons can create insertions, deletions, or inversions in the genome. In some embodiments, the methods, compositions, and cells provided herein do not involve the use of transposons (e.g., "non-transposon-mediated").

[0184] In one embodiment, the disclosure provides a modified chromosome comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the disclosure provides a modified cell comprising a modified chromosome comprising a modified chromosome comprising a modified chromosome comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In another embodiment, the disclosure provides a modified cell comprising a modified chromosome comprising a modified chromosome comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof.

[0185] In one embodiment, the disclosure provides a product comprising a modified chromosome containing a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the disclosure provides a product comprising a modified plant or a portion thereof containing a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the disclosure provides a product comprising a modified plant cell containing a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the disclosure provides a product comprising a modified cell containing a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of the gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene or a portion thereof. In one embodiment, the product comprises silage, flour, cellulose, sugars, starches, fats, syrups, or proteins derived from plants, plant parts, or plant cells.

[0186] In one embodiment, the disclosure provides a modified cell comprising a) a non-transposon-mediated genomic deletion of at least one gene or a portion thereof at an endogenous locus of at least one gene, or b) a non-transposon-mediated and non-T-DNA-mediated insertion of a polynucleotide sequence into at least one gene, wherein the deletion or insertion produces a dominant-positive allele of at least one gene. In one embodiment, the insertion comprises a regulatory element. In another embodiment, the regulatory element is selected from the group consisting of promoter sequences, transcription start site sequences, transcription termination site sequences, enhancer sequences, and designed elements.

[0187] In another aspect, the disclosure provides a modified cell comprising a non-transposon-mediated genomic deletion or inversion of at least one gene or a portion thereof at an endogenous locus of at least one gene, wherein the deletion or inversion produces a dominant-negative allele of at least one gene. In yet another aspect, the disclosure provides a modified cell comprising a non-transposon-mediated genomic deletion or inversion of a gene or a portion thereof at an endogenous locus of at least one gene, wherein the deletion or inversion results in the production of an RNA transcript containing a sequence complementary to the native transcript sequence of the gene. In one aspect, the disclosure provides a modified cell comprising targeted editing of at least one gene or a portion thereof, wherein the targeted editing produces an RNA transcript complementary to the native transcript sequence of the gene. In one aspect, the RNA transcript is a complete antisense transcript. In another aspect, the RNA transcript is a partial antisense transcript. In a further aspect, the RNA transcript is a partial sense transcript. In yet another embodiment, the RNA transcript is a complete sense transcript. In yet another embodiment, the RNA transcript is a native transcript of the gene. In yet another embodiment, the native transcript of the gene is a partial or complete sense transcript.

[0188] The dominant allele of a gene region can also be created by inducing constitutive expression of the gene region by inserting a designed element into the promoter of the gene region.

[0189] In one embodiment, the Disclosure provides a method for modifying gene expression, comprising: a) inducing a double-strand break at a target site of a gene using a targeted editing technique; b) inserting a donor sequence at the double-strand break, wherein the donor sequence comprises a designed element capable of inducing increased or ectopic expression of the gene; and c) identifying at least one cell containing the donor sequence insertion, wherein gene expression is increased in at least one tissue compared to control cells that do not contain the donor sequence insertion.

[0190] In another embodiment, the Disclosure provides a method for a) providing one or more cells to at least one RNA guide nuclease and at least one donor molecule, or one or more vectors encoding at least one RNA guide nuclease and at least one donor molecule, wherein the at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site of at least one gene, the donor molecule comprises a designed element, the RNA guide nuclease produces a double-strand break at the target site, and the donor molecule is inserted by the double-strand break; and b) identifying one or more cells containing the insertion of the donor molecule at the target site; and c) selecting one or more cells containing the insertion of the donor molecule at the target site.

[0191] In one embodiment, the target site is located downstream of a TATA box upstream of a gene. In another embodiment, the target site is located upstream of a TATA box upstream of a gene. In yet another embodiment, the target site is located downstream of a TATA box operably linked to at least one gene. In one embodiment, the target site is located within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 90, 1000, 1500, 2000, 2500, or 5000 nucleotides from a TATA box operably linked to at least one gene. In yet another embodiment, the target site is located at 10-5000, 10-2500, 10-1500, 10-1000, 10-750, 10-500, 10-250, 10-100, 20-100, 20-250, 20-500, or 50-500 nucleotides from a TATA box operably linked to at least one gene. In yet another embodiment, the target site is located within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 5000 nucleotides from the gene promoter. In yet another embodiment, the target site is located upstream of the upstream initiator element of the gene. In yet another embodiment, the target site is located downstream of the upstream initiator element of the gene.In yet another aspect, the target site is located within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 5000 nucleotides from the initiator element of the gene.

[0192] In one embodiment, a “TATA box” comprises the core DNA sequence 5'-TATAAA-3' or a variant thereof and is frequently associated with promoters of eukaryotic genes. Typically, TATA boxes are located approximately 25–35 nucleotides upstream from the transcription start site of a gene, but this is not always the case. TATA boxes often function as binding sites for enabling the expression of a gene operably ligated by a transcription factor, or for inhibiting the expression of a gene operably ligated by a histone. In one embodiment, a TATA box is an initiator element. An initiator element is a core promoter that facilitates the binding of a transcription factor and enhances the expression of an operably ligated gene. In one embodiment, the initiator sequence provided herein comprises the sequence 5'-[C / T][C / T]AN[A / T][C / T][C / T]-3'.

[0193] Dominant-negative alleles can also be created by editing the genome to include a tissue-specific or tissue-preferential promoter in the gene region so that the tissue-specific or tissue-preferential promoter is oriented in the opposite direction to the targeted gene. For example, by reversing the orientation of the tissue-specific promoter downstream of the 3'-UTR of the gene region, the tissue-specific promoter can generate a complete antisense gene region RNA transcript. While not bound by either theory, antisense gene region RNA transcripts expressed by antisense tissue-specific promoters can suppress gene region expression in a tissue-specific manner.

[0194] In one embodiment, the Disclosure provides a method for reducing gene expression in at least one cell, comprising: a) inducing a double-strand break at a target site of the gene using targeted editing techniques; b) inserting a donor sequence at the double-strand break, wherein the donor sequence includes a tissue-specific or tissue-preferential promoter and is inserted at the target site such that the tissue-specific or tissue-preferential promoter is reversed relative to the gene; and c) identifying at least one cell containing the reversed donor sequence insertion, wherein gene expression is reduced compared to a control cell without the donor sequence insertion. In one embodiment, the method provided herein further comprises removing the native promoter of the gene using targeted editing techniques. As used herein, “native promoter” means a promoter that produces a sense mRNA transcript of an operablely linked gene.

[0195] In another embodiment, the Disclosure provides a method for a) providing one or more cells to an insertion of a donor molecule at a target site such that the tissue-specific or tissue-preferential promoter is reversed relative to the gene, and the insertion of the donor molecule at a target site such that the tissue-specific or tissue-preferential promoter is reversed relative to the gene, and the insertion of the donor molecule at a target site such that the insertion of the donor molecule at a target site is reversed relative to the gene.

[0196] In one embodiment, the target site is located downstream of the gene's 3'-UTR. In another embodiment, the target site is located within the gene's 3'-UTR. In yet another embodiment, the target site is located within an intron of a gene. In yet another embodiment, the target site is located within an exon of a gene. In one embodiment, the target site is located with the gene's 5'-UTR. In another embodiment, the target site is located upstream of the gene's 5'-UTR. In yet another embodiment, the target site is located within the gene's promoter.

[0197] In one embodiment, the donor molecule comprises a polynucleotide encoding a promoter. In another embodiment, the donor molecule comprises a polynucleotide encoding a promoter selected from the group consisting of tissue-specific promoters, tissue-preferential promoters, constitutive promoters, and inducible promoters. In yet another embodiment, the donor molecule provided herein comprises a polynucleotide encoding a tissue-specific or tissue-preferential promoter. In yet another embodiment, the donor molecule provided herein comprises a polynucleotide encoding a constitutive promoter. In yet another embodiment, the donor molecule provided herein comprises a polynucleotide encoding an inducible promoter.

[0198] In one embodiment, a tissue-specific or tissue-preferential promoter is selected from the group consisting of leaf-specific promoters, leaf-preferential promoters, stem-specific promoters, stem-preferential promoters, vascular bundle-specific promoters, vascular bundle-preferential promoters, root-specific promoters, root-preferential promoters, inflorescence-specific promoters, inflorescence-preferential promoters, pollen-specific promoters, pollen-preferential promoters, anther-specific promoters, anther-preferential promoters, ovule-specific promoters, ovule-preferential promoters, seed-specific promoters, seed-preferential promoters, embryo-specific promoters, embryo-preferential promoters, endomilk-specific promoters, endomilk-preferential promoters, pericarp-specific promoters, pericarp-preferential promoters, alleuron-specific promoters, alleuron-preferential promoters, meristem-specific promoters, meristem-preferential promoters, fruit-specific promoters, fruit-preferential promoters, pod-specific promoters, pod-preferential promoters, epidermis-specific promoters, epidermis-preferential promoters, mitochondrial-specific promoters, mitochondrial-preferential promoters, chloroplast-specific promoters, and chloroplast-preferential promoters. In another embodiment, a tissue-specific or tissue-preferential promoter provided herein is an RTBV promoter. In one embodiment, the tissue-specific or tissue-preferential promoters provided herein express the antisense mRNA transcript of a gene.

[0199] Targeted editing techniques can be used to insert donor molecules into target sites of genomic loci. When a donor molecule containing a non-coding RNA target site is inserted into the 5'-UTR, exon, intron, or 3'-UTR of the gene of interest, RNA transcription or protein translation of the gene of interest can be repressed by complementary non-coding RNA. If the gene of interest is targeted by non-coding RNA (e.g., miRNA or siRNA), the cleaved mRNA of the gene of interest can generate secondary siRNA, which can further repress the transcription or translation of the gene of interest. Since secondary siRNA is complementary to the allele with or without the insertion of the non-coding RNA target site, such secondary repression can act in a dominant manner.

[0200] In one embodiment, an engineered or artificial miRNA is created to target a native gene region. In another embodiment, the gene region is edited to be complementary to the native miRNA. The engineered miRNA is useful for repressing the targeted gene with increased specificity. See, for example, Parizotto et al., Genes Dev. 18:2237-2242 (2004), and U.S. Patent Applications Publications 2004 / 0053411, 2004 / 0268441, 2005 / 0144669, and 2005 / 0037988. The contents and disclosures of these are incorporated herein by reference. miRNA is a non-protein-coding RNA. When a miRNA precursor molecule is cleaved, a mature miRNA is formed, typically about 19–25 nucleotides long (generally about 20–24 nucleotides in plants), e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides long, containing sequences corresponding to the gene targeted for repression and / or its complement. The mature miRNA hybridizes to the target mRNA transcript, guiding the binding of the protein complex to the target transcript, which functions to inhibit translation and / or degrade the transcript, thus negatively regulating or repressing the expression of the targeted gene. In plants, miRNA precursors are also useful in directing the in-phase production of siRNA, trans-acting siRNA (ta-siRNA), in processes requiring RNA-dependent RNA polymerase to induce repression of the target gene. See, for example, Allen et al., Cell 121:207-221 (2005), Vaucheret Science STKE, 2005:pe43 (2005), and Yoshikawa et al. Genes Dev., 19:2164-2175 (2005). The contents and disclosures of these are incorporated herein by reference.

[0201] Plant miRNAs regulate their target genes by recognizing and binding to nearly perfectly complementary sequences (miRNA recognition sites) in target transcripts, followed by cleavage of the transcript by RNase III enzymes such as Argonaut 1. In plants, certain mismatches between a given miRNA recognition site and the corresponding mature miRNA, particularly mismatched nucleotides at positions 10 and 11 of the mature miRNA, are unacceptable. The positions within the mature miRNA are indicated in the 5' to 3' direction. Typically, complete complementarity between the given miRNA recognition site and the corresponding mature miRNA is required at positions 10 and 11 of the mature miRNA. See, for example, Franco-Zorrilla et al. (2007) Nature Genetics, 39:1033-1037, and Axtell et al. (2006) Cell, 127:565-577.

[0202] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase," available online at microrna.sanger.ac.uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to arise in intergeneric regions within the genome, both in isolation and in clusters, but may also be located entirely or partially within the introns of other genes (both protein-coding and non-protein-coding). For a recent overview of miRNA synthesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes can occur, at least in some cases, under the promotional control of the MIR gene's own promoter. The primary transcript, called "pri-miRNA," can be quite large (several kilobases) and may be polycistronic, containing one or more pre-miRNAs (foldback structures containing stem-loop sequences that are processed into mature miRNAs) as well as the mRNA's usual 5' "cap" and polyadenylated tail. See, for example, Figure 1 in Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385.

[0203] Transgenic expression of miRNAs (whether naturally occurring or artificial sequences) can be used to regulate the expression of miRNA target genes(s). Recognition sites of miRNAs have been identified in all regions of mRNA, including the 5' untranslated region, coding region, and 3' untranslated region, demonstrating that the location of the miRNA target site relative to the coding sequence does not necessarily affect repression (see, for example, Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290, and Sunkar and Zhu (2004) Plant Cell, 16:2001-2019). Because miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. MIR gene promoters can have highly specific expression patterns (e.g., cell-specific, tissue-specific, time-specific, or inducible), making them useful in recombinant constructs that induce such specific transcription of operably linked DNA sequences. The various uses of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication No. 2006 / 0200878A1, incorporated herein by reference. Non-limiting examples of these uses include (1) expression of native miRNA or miRNA precursor sequences that repress target genes, (2) expression of artificial miRNA or miRNA precursor sequences that repress target genes, (3) expression of transgenes having miRNA recognition sites that are repressed when mature miRNA is expressed, and (4) expression of transgenes driven by miRNA promoters.

[0204] As demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333, the design of artificial miRNA sequences can be as simple as substituting a sequence complementary to the intended target of nucleotides in the miRNA stem region of a miRNA precursor. One non-limiting example of a common method for determining nucleotide changes in a native miRNA sequence to produce an engineered miRNA precursor includes the following steps: (a) Select a unique target sequence of at least 18 nucleotides specific to the target gene by using a sequence alignment tool such as BLAST (see, e.g., Altschul et al. (1990) J.Mol.Biol., 215:403-410, Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) from both tobacco cDNA and genomic DNA databases, to identify any possible matches with target transcript orthologues and unrelated genes, thereby avoiding unintended silencing of non-target sequences; (b) Analyze the target gene for unwanted sequences (e.g., matches with sequences from non-target species), and determine the GC content of each possible 19-mer segment, Reynolds score (Reynolds et al. (2004) Nature Scoring functional asymmetry characterized by a Reynolds score > 4, a GC content of about 40% to 60%, a negative ΔΔG, a negative GC content, a negative GC content, a negative GC content, a negative GC content, a negative ΔΔGThe position of every three nucleotides in siRNA has been reported to be particularly important in affecting the effectiveness of RNAi, and an algorithm called "siExplorer" is publicly available at rna.chem.tu-tokyo.ac.jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complement of a selected 19-mer for use in the production of a modified mature miRNA [the additional nucleotide at position 20 is preferably matched to a selected target sequence, and the nucleotide at position 21 is preferably selected to be unpaired to prevent the spread of silencing to the target transcript, or to be paired with the target sequence to enhance the spread of silencing to the target transcript]; and (d) transforming plants with the artificial miRNA.

[0205] The siRNA pathway involves non-stepwise cleavage of longer double-stranded RNA intermediates (RNA double helix) into small interfering RNA (siRNA). siRNA size or length ranges from about 19 to about 25 nucleotides or base pairs, although a common class of siRNAs includes those containing 21 or 24 base pairs. Therefore, the transcriptable DNA sequences or repression elements of this application may encode RNA molecules with a length of at least about 19 to about 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0206] In the ta-siRNA pathway, miRNAs help guide the in-phase processing of the siRNA primary transcript in a process that requires RNA-dependent RNA polymerase for the production of a double-stranded RNA precursor. Ta-siRNAs are defined by the absence of secondary structure, the miRNA target site that initiates double-stranded RNA production, the need for DCL4 and RNA-dependent RNA polymerase (RDR6), and the production of multiple approximately 21nt small RNAs with perfectly matched double helixes and completely identical phases, accompanied by a 2-nucleotide 3' overhang (see Allen et al. (2005) Cell, 121:207-221). The size or length of ta-siRNAs ranges from approximately 20 to approximately 22 nucleotides or base pairs, but is most often 21 base pairs. Thus, the donor molecules or vectors of this application may encode RNA molecules that are at least approximately 20 to approximately 22 nucleotides in length, for example, 20, 21, or 22 nucleotides. The donor molecules and vectors provided herein may include ta-siRNA scaffolds. For a suitable method for constructing a ta-siRNA scaffold, see U.S. Patent No. 9,309,512, which is incorporated herein by reference in whole.

[0207] This disclosure provides a method for generating a dominant-negative allele of a gene, comprising using targeted editing techniques to introduce at least one non-coding RNA target site into the gene. In one embodiment, the dominant-negative allele of the gene is downregulated compared to an allele of the gene that does not contain at least one non-coding RNA target site. In another embodiment, a secondary siRNA complementary to the gene is generated. In another embodiment, the at least one non-coding RNA target site is a miRNA target site or an siRNA target site. In a further embodiment, the at least one non-coding RNA target site is introduced into a region of the gene selected from the group consisting of the 5'-UTR, exons, introns, and 3'-UTR. In another embodiment, the at least one non-coding RNA target site is introduced into an exon of the gene. In another embodiment, the at least one non-coding RNA target site is introduced into an intron of the gene. In another embodiment, the at least one non-coding RNA target site is introduced into the 5'-UTR of the gene. In yet another embodiment, the at least one non-coding RNA target site is introduced into the 3'-UTR of the gene.

[0208] In another aspect, the disclosure provides a modified cell comprising a non-transgenic dominant-negative allele of a gene, wherein the dominant-negative allele comprises a heterologous non-coding RNA target site at an endogenous locus of the gene.

[0209] Dominant alleles can also be created by editing the allele of a protein-coding gene region to produce a cleaved protein, which interferes with the activity of the wild-type protein and results in a dominant effect. In one embodiment, a dominant-positive allele is created by introducing targeted editing to a protein-coding gene to produce a cleaved protein. In one embodiment, a dominant-negative allele is created by introducing targeted editing to a protein-coding gene to produce a cleaved protein. In one embodiment, the cleaved proteins provided herein interfere with protein-protein binding, DNA-protein binding, or RNA-protein binding. In one embodiment, the cleaved proteins provided herein are microproteins. As used herein, a microprotein refers to a protein approximately 100-200 amino acids long that codes only for protein-protein interaction or binding domains (see, for example, Seo et al., Trends in Plant Sciences, 2011, 10:541-549). Microproteins often arise from functional genes that have undergone mutations that cause the loss of a functional protein domain. In one embodiment, the microprotein is at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 225 amino acids long. In one embodiment, the microprotein inhibits the activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proteins in a cell. In another embodiment, the microprotein promotes the activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proteins in a cell. The microprotein may compete with the second protein for a binding site in the third protein.In one embodiment, the microprotein may prevent the second protein from binding to the third protein, resulting in inhibition of its activity. Alternatively, in another embodiment, the microprotein may bind to the third protein instead of the second protein, thereby promoting the activity of the third protein.

[0210] In one embodiment, a plant containing a dominant-negative allele encoding a microprotein is subject to improvement of traits selected from the group consisting of flowering time, meristem size, insect resistance, herbicide tolerance, and shade avoidance. In one embodiment, a plant containing a dominant-positive allele encoding a microprotein is subject to improvement of traits selected from the group consisting of flowering time, meristem size, insect resistance, herbicide tolerance, and shade avoidance.

[0211] In one embodiment, the cleaved proteins provided herein are selected from the group consisting of cleaved CLAVATA proteins, cleaved CORYNE proteins, cleaved BAM receptors, cleaved receptor-like protein kinase 2 (RPK2) proteins, and cleaved G protein beta subunit 1 (AGB1) proteins. In another embodiment, the CLAVATA proteins provided herein are CLAVATA1 proteins, CLAVATA2 proteins, or CLAVATA3 proteins.

[0212] In one embodiment, the present disclosure provides a method for generating a dominant-negative allele of a gene, comprising: a) inducing a double-strand break in the genome of at least one cell using a targeted editing technique at a target site of the gene, wherein the double-strand break is repaired by non-homologous end joining; and b) identifying at least one cell containing an insertion or deletion at the target site, wherein the insertion or deletion at the target site results in the generation of a dominant-negative allele of the gene.

[0213] In yet another embodiment, the disclosure provides a modified cell comprising at least one insertion or deletion at an endogenous locus of at least one gene generated by a targeted editing technique, wherein the insertion or deletion results in the expression of a cleavage protein.

[0214] In another embodiment, the Disclosure provides a method comprising: a) providing one or more cells to at least one RNA guide nuclease, or one or more vectors encoding at least one RNA guide nuclease, such that the at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site of at least one gene, and the RNA guide nuclease produces a double-strand break at the target site; b) identifying at least one cell containing an insertion or deletion at the target site, such that the insertion or deletion at the target site results in the generation of a dominant-negative allele of at least one gene; and c) selecting one or more cells containing a dominant-negative allele of at least one gene.

[0215] The disclosure also provides a method for generating a dominant allele of a gene, which involves using targeted editing techniques to introduce nonsense mutations into a gene to produce a cleaved protein. In one embodiment, the cleaved protein is a microprotein. In one embodiment, the targeted editing technique includes deletions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 nucleotides. In one embodiment, the targeted editing technique includes insertions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 nucleotides. In one embodiment, the targeted editing technique includes inversions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 nucleotides.

[0216] In one embodiment, a dominant-positive allele is generated by introducing targeted editing into a protein-coding gene to produce a cleavage protein. In one embodiment, the present disclosure provides a method for generating a dominant-positive allele of a gene, comprising: a) inducing a double-strand break in the genome of at least one cell using targeted editing techniques at a target site of the gene, wherein the double-strand break is repaired by non-homologous end joining; and b) identifying at least one cell containing an insertion or deletion at the target site, wherein the insertion or deletion at the target site results in the generation of a dominant-positive allele of the gene.

[0217] In another embodiment, the Disclosure provides a method comprising: a) providing one or more vectors to one or more cells, wherein the one or more vectors comprise at least one polynucleotide encoding at least one RNA guide nuclease, the at least one RNA guide nuclease capable of binding to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides at a target site of at least one gene, the RNA guide nuclease producing a double-strand break at the target site, and the double-strand break being repaired by non-homologous end joining; b) identifying at least one cell containing an insertion or deletion at a target site, the insertion or deletion at the target site resulting in the generation of a dominant-positive allele of at least one gene; and c) selecting one or more cells containing a dominant-positive allele of at least one gene.

[0218] In one embodiment, the insertions or deletions provided herein disrupt an intron / exon splice site. An intron / exon splice site refers to the boundary between an intron and an exon within a gene. In eukaryotes, introns are typically processed from the RNA transcript by the spliceosome so that an mRNA transcript containing only the exon sequence is produced, but this is not always the case. If an intron / exon splice site is disrupted, the spliceosome may not be able to properly remove the intron sequence, potentially resulting in a protein with one or more nonsense mutations that produce an immature stop codon. In one embodiment, the nonsense mutations produce a cleaved protein. In one embodiment, the cleaved protein contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 fewer amino acids than the endogenous protein encoded by the gene lacking the nonsense mutation.

[0219] In one embodiment, a nonsense mutation is a mutation that results in an immature stop codon in transcribed mRNA. In another embodiment, the insertions or deletions provided herein are located within an exon. In another embodiment, the insertions or deletions provided herein are located within an intron. In another embodiment, the insertions or deletions provided herein are located within a 5'-UTR or a 3'-UTR. In one embodiment, the insertions or deletions provided herein are located within a structure selected from the group consisting of intron / exon splice sites, exons, introns, 5'-UTRs, and 3'-UTRs. In yet another embodiment, the dominant-negative alleles provided herein include one or more, two or more, three or more, four or more, or five or more insertions and / or deletions. In yet another embodiment, the dominant-positive alleles provided herein include one or more, two or more, three or more, four or more, or five or more insertions and / or deletions.

[0220] In another embodiment, the nonsense mutations provided herein are located within an exon. In one embodiment, the insertions or deletions provided herein are located within a structure selected from the group consisting of an intron / exon splice site and an exon. The insertions or deletions provided herein can produce proteins having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nonsense mutations.

[0221] In one embodiment, the dominant-negative allele provided herein comprises a polynucleotide containing an immature stop codon compared to the polynucleotide of a control allele. The immature stop codon is a stop codon located upstream of the normal stop codon of a gene. The immature stop codon generates a cleaved protein. A stop codon is a nucleotide triplet in mRNA that signals the end of protein translation from mRNA. In one embodiment, the dominant-negative allele provided herein comprises a polynucleotide encoding a cleaved protein. In one aspect, the cleaved proteins provided herein are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 400, or at least 500 amino acids shorter than the full-length protein. In one embodiment, the cleaved proteins provided herein are generated by the insertion or deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1000, at least 1500, or at least 2000 nucleotides.

[0222] In one embodiment, the dominant-positive allele provided herein comprises a polynucleotide containing an immature stop codon compared to the polynucleotide of a control allele. The immature stop codon is a stop codon located upstream of the normal stop codon of a gene. The immature stop codon generates a cleaved protein. A stop codon is a nucleotide triplet in mRNA that signals the end of protein translation from mRNA. In one embodiment, the dominant-positive allele provided herein comprises a polynucleotide encoding a cleaved protein. In one aspect, the cleaved proteins provided herein are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 400, or at least 500 amino acids shorter than the full-length protein. In one embodiment, the cleaved proteins provided herein are generated by the insertion or deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1000, at least 1500, or at least 2000 nucleotides.

[0223] This disclosure provides a method for generating a dominant-negative allele of a gene in a cell, comprising deleting a portion of the gene using a targeted editing technique, wherein a microprotein is generated after the deletion of the portion of the gene. In one embodiment, the cleaved protein is a microprotein. In another embodiment, the dominant-negative allele provided herein encodes a microprotein. In a further embodiment, the dominant-positive allele provided herein encodes a microprotein. As used herein, “microprotein” means a short single-domain protein having the ability to interfere with a larger multi-domain protein. In one embodiment, the microprotein provided herein interferes with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten other proteins. In one embodiment, the microprotein provided herein can prevent the second protein from binding to a nucleic acid molecule. In another embodiment, the microprotein provided herein can prevent the second protein from binding to a third protein. The third protein may or may not be identical to the second protein. In another embodiment, the microproteins provided herein can bind to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten other proteins. In one embodiment, the microproteins provided herein can form heterodimers with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten other proteins. In another embodiment, the microproteins provided herein can form homodimers.As used herein, “homodimer” refers to the hybridization or binding of two identical molecules (e.g., protein A and protein A), and “heterodimer” refers to the hybridization or binding of two different macromolecules (e.g., protein A and protein B; protein A and DNA; protein A and RNA).

[0224] Members of the pentatricopeptide repeat (PPR) gene family are common in plant genomes. Many PPR proteins can bind to RNA molecules in a sequence-specific manner. A PPR protein contains 2 to 30 PPR motifs, each of which aligns with a single nucleotide within the RNA molecule. Within a PPR motif, the presence of two or three specific amino acids confers nucleotide specificity. For example, but are not limited to, when threonine is at position 6 and asparagine is at position 1', the PPR motif binds to an adenine nucleotide; when threonine is at position 6 and aspartic acid is at position 1', the PPR motif binds to a guanine nucleotide; when asparagine is at position 6 and aspartic acid is at position 1', the PPR motif binds to a uracil (or thymine) nucleotide; and when asparagine is at position 6 and asparagine or serine is at position 1', the PPR motif binds to a cytosine nucleotide.

[0225] While not limited to these, engineered PPR proteins can be generated by at least two construction strategies. In the first strategy, the PPR protein is constructed by treating each PPR motif as a separate block, so that the PPR protein is constructed by arranging multiple desired motifs. Thus, the resulting engineered PPR protein can bind to a target RNA molecule. However, this strategy does not always work because each PPR motif contains an internal scaffold between the 1' and 6' positions, and this internal motif scaffold is not shared between different PPR proteins. The second strategy utilizes an existing internal motif scaffold. In the second strategy, site-directed mutagenesis at the 1' and 6' positions is used to edit an existing PPR protein to make it specific to a new target RNA molecule.

[0226] As used herein, "engineered PPR protein" and "engineered PPR motif" refer to synthetically produced PPR proteins or PPR motifs that do not exist in nature and are capable of site-specifically binding to RNA sequences.

[0227] This disclosure provides a method comprising: a) providing cells with an engineered PPR protein, or a vector encoding an engineered PPR protein operably linked to a promoter, such that the engineered PPR protein can bind to an RNA transcript of a target gene; b) selecting one or more cells expressing the engineered PPR protein from step (a); and c) identifying one or more cells selected in step (b) that exhibit altered expression of the target gene. In one embodiment, the engineered PPR protein can bind to at least one non-coding RNA target site of the RNA transcript. In one embodiment, the engineered PPR protein binds to at least one non-coding RNA target site of the RNA transcript. In one embodiment, altered expression is increased expression. In another embodiment, altered expression is reduced expression. In one embodiment, the promoter is the native promoter of the target gene. In another embodiment, the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, a tissue-preferential promoter, and an inducible promoter.

[0228] In one embodiment, the engineered PPR protein provided herein binds to a non-coding RNA target site of a target RNA molecule, preventing the non-coding RNA from cleaving the target RNA or inhibiting the translation of the target RNA. In another embodiment, the engineered PPR protein provided herein is directed toward the degradation of the target RNA molecule. In one embodiment, the engineered PPR protein provided herein comprises at least one RNA nuclease domain. In another embodiment, the RNA nuclease domain provided herein is an NYN nuclease domain or an SMR (small MutS-related) domain.

[0229] In one embodiment, the manipulated PPR protein or manipulated PPR motif provided herein binds to at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 nucleotides of an RNA molecule. In another aspect, the engineered PPR proteins or engineered PPR motifs provided herein bind to 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 15-30 nucleotides of an RNA molecule.

[0230] In one embodiment, the engineered PPR protein provided herein can act as a dominant-negative allele. In another embodiment, the engineered PPR protein provided herein can act as a dominant-positive allele.

[0231] In one embodiment, the engineered PPR protein targets mitochondria or chloroplasts. In another embodiment, the engineered PPR protein targets the nucleus. In yet another embodiment, the engineered PPR protein targets the cytoplasm of a cell. While not limited to any of these theories, the target of a protein can be made to a specific cellular structure by adding or editing a transport peptide at the N-terminus of the protein.

[0232] In one embodiment, the genome editing system provided herein includes tgOligo as a tether molecule. In another embodiment, the tether molecule is a crosslinking agent coupled to a nuclease or a DNA targeting guide molecule. In a further embodiment, the tether molecule is a dimerizing domain coupled to a nuclease.

[0233] In one embodiment, a tether molecule can link two or more DNA binding mechanisms bound to two genomic loci. In another embodiment, a tether molecule can link two or more DNA binding mechanisms bound to two genomic loci located within a single chromosome that flank a target genomic region. In yet another embodiment, a tether molecule can link two or more DNA binding mechanisms bound to two genomic loci located on separate chromosomes.

[0234] In one embodiment, the present disclosure is a method for generating a dominant-negative allele of at least one gene in at least one cell, comprising: a) introducing into at least one cell a genome editing system comprising: i) a site-specific nuclease or a molecule encoding a site-specific nuclease; ii) an sgRNA or a molecule encoding an sgRNA; and iii) one or more molecules encoding at least a first tether-guide oligo (tgOligo) and a second tgOligo, or the first and second tgOligo, operably linked to at least one promoter; and b) generating a first double-strand break and a second double-strand break in at least one gene, wherein the first tgOli The present invention provides a method comprising: a) the generation of a dominant-negative allele of a gene encoding a cleavage protein, wherein go and a second tgOligo hybridize to the 3' free ends of opposing strands in the first and second double-strand breaks, resulting in the deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 750, at least 1000, at least 2500, or at least 5000 nucleotides of at least one gene, thereby generating a dominant-negative allele of a gene encoding a cleavage protein; and c) identifying and selecting at least one cell containing a cleavage protein.

[0235] In one embodiment, the present disclosure is a method for generating a dominant-negative allele of at least one gene in at least one cell, comprising: a) introducing into at least one cell a genome editing system comprising: i) a site-specific nuclease or a molecule encoding a site-specific nuclease; ii) an sgRNA or a molecule encoding an sgRNA; and iii) one or more molecules encoding at least a first tether-guide oligo (tgOligo) and a second tgOligo, or the first and second tgOligo, operably linked to at least one promoter; and b) generating a first double-strand break and a second double-strand break in at least one gene. The present invention provides a method comprising: a) the generation of a dominant-negative allele of a gene encoding a cleavage protein, wherein a first tgOligo and a second tgOligo hybridize to the 3' free ends of opposing strands in the first and second double-strand breaks, resulting in the deletion of 1 to 5000, 5 to 5000, 10 to 5000, 25 to 2500, 25 to 1000, 25 to 750, 25 to 500, 25 to 100, 50 to 5000, 50 to 1000, 50 to 500, 100 to 1000, or 1000 to 5000 nucleotides of at least one gene, thereby generating a dominant-negative allele of a gene encoding a cleavage protein; and c) identifying and selecting at least one cell containing a cleavage protein.

[0236] In another embodiment, the present disclosure provides a method for generating a dominant-negative allele of at least one gene in at least one cell, comprising: a) introducing one or more vectors into at least one cell that encode at least a first tgOligo and a second tgOligo operably ligated to i) at least one site-specific nuclease, ii) at least one sgRNA, and iii) at least one promoter; and b) generating a first double-strand break and a second double-strand break in the gene. The present invention provides a method comprising: a) the first tgOligo and the second tgOligo hybridize to the 3' free ends of the opposing strands in the first and second double-strand breaks, causing the region of at least one gene between the first and second double-strand breaks to be oriented in the opposite direction, thereby generating a dominant-negative allele of at least one gene encoding the antisense RNA transcript of the gene; and c) identifying and selecting at least one cell containing the antisense RNA transcript of at least one gene.

[0237] As used herein, "tether-guided oligo" (tgOligo) refers to an oligonucleotide containing a sequence segment that can hybridize to the 3' free end (also called the 3' free flap) of the non-target strand of a double-stranded DNA molecule that is recognized and cleaved by a CRISPR gRNA-Cas complex. When a tgOligo recognizes and hybridizes to the 3' free end of the non-target strand of a gRNA's target site, this tgOligo corresponds to that gRNA. A tgOligo may be a DNA molecule, an RNA molecule, or a mixture of nucleotides. A hybrid tgOligo is a tgOligo that can recognize and hybridize to the non-target 3' free end produced by two separate CRISPR gRNA-Cas complexes.

[0238] As used herein, "tether guide RNA" (tgRNA) refers to an RNA molecule that contains both a guide RNA (gRNA) sequence and a tether RNA sequence, and in which the tether RNA sequence can hybridize with a desired genomic site (a site called a "tether site").

[0239] In one embodiment, the methods provided herein involve the use of one or more tgOligos, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. In one embodiment, tgOligo is a DNA molecule. In another embodiment, tgOligo is an RNA molecule. In a further embodiment, tgOligo is a mixture of DNA and RNA molecules. In one embodiment, tgOligo is single-stranded. In another embodiment, tgOligo is double-stranded. In one embodiment, at least one or at least two tgOligos are used simultaneously with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. In another embodiment, at least one tgOligo is not used simultaneously with a site-specific nuclease. In one embodiment, at least one or at least two tgOligos are linked to at least one or at least two Cas9 proteins. In one embodiment, a first tgOligo is linked to a first Cas9 protein, and a second tgOligo is linked to a second Cas9 protein. In another embodiment, at least one or at least two tgOligos are linked to at least one or at least two inactivated Cas9 proteins.

[0240] In yet another embodiment, the tgOligo provided herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, or at least 25,000 nucleotides. In a further embodiment, the tgOligo provided herein includes 5 to 25,000 nucleotides, 5 to 10,000 nucleotides, 5 to 5,000 nucleotides, 20 to 10,000 nucleotides, 20 to 5,000 nucleotides, 20 to 1,000 nucleotides, 20 to 500 nucleotides, 20 to 250 nucleotides, 50 to 2,500 nucleotides, 50 to 1,000 nucleotides, 50 to 500 nucleotides, 50 to 250 nucleotides, 100 to 2,500 nucleotides, 100 to 1,000 nucleotides, 100 to 500 nucleotides, or 1,000 to 10,000 nucleotides.

[0241] In one embodiment, the first tgOligo and the second tgOligo are complementary to each other by at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%. In one embodiment, the first tgOligo and the second tgOligo are complementary to each other by at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, and a small number. For at least 125, at least 150, at least 175, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 nucleotides, they are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% complementary with one another. In one embodiment, the first tgOligo includes a sense strand, and the second tgOligo includes an antisense strand.

[0242] In one embodiment, tgOligo is provided to cells. In another embodiment, tgOligo is encoded by a vector. In yet another embodiment, the site-specific nuclease and tgOligo are encoded by a single vector. In yet another embodiment, the site-specific nuclease and tgOligo are encoded by two or more vectors.

[0243] The methods provided herein are suitable for generating dominant alleles of protein-coding genes and non-coding RNAs. Examples of target genes in plant genomes envisioned by this disclosure include, but are not limited to, genes for resistance to diseases, insects, or pests; herbicide resistance; quality improvements, such as yield, nutrient enhancement, environmental tolerance, or stress tolerance; or starch production (U.S. Patents No. 6,538,181, 6,538,179, 6,538,178, 5,750,876, and 6,47) (Patent No. 6,295); Production volume of modified oil (Patent Nos. 6,444,876, 6,426,447, and 6,380,462); High oil production volume (Patent Nos. 6,495,739, 5,608,149, 6,483,008, and 6,476,295); Modified fatty acid content (Patent Nos. 6,828,475, 6,822,141, and 6,770,465) (US Patent Nos. 6,706,950, 6,660,849, 6,596,538, 6,589,767, 6,537,750, 6,489,461, 6,459,018); high protein production (US Patent No. 6,380,466); fruit ripening (US Patent No. 5,512,466); nutritional enhancement for animals and humans (US Patent Nos. 6,723,837, 6,65 This includes genes for any desirable changes in the physiology, growth, development, morphology or plant products of plants, including U.S. Patent Nos. 3,530, 6,5412,59, 5,985,605, and 6,171,640; or biomacromolecules (U.S. Patent Nos. RE37,543, 6,228,623, 5,958,745, and U.S. Patent Publication No. US20030028917).Furthermore, environmental stress resistance (U.S. Patent No. 6,072,103); pharmaceutical peptides and secretable peptides (U.S. Patent Nos. 6,812,379, 6,774,283, 6,140,075, and 6,080,560); improved processing properties (U.S. Patent No. 6,476,295); improved digestibility (U.S. Patent No. 6,531,648); low raffinose (U.S. Patent No. 6,166,292); industrial Enzyme production (U.S. Patent No. 5,543,576); improved flavor (U.S. Patent No. 6,011,199); nitrogen fixation (U.S. Patent No. 5,229,114); hybrid seed production (U.S. Patent No. 5,689,041); fiber production (U.S. Patents No. 6,576,818, 6,271,443, 5,981,834, and 5,869,720); and biofuel production (U.S. Patent No. 5,998,700).

[0244] In one embodiment, the gene edited by the method provided herein is selected from the group consisting of the Y1 gene, the brachytic2 gene, the GA3 oxidase gene, and the GA20 oxidase gene. In another embodiment, the gene edited by the method provided herein encodes non-coding RNA. In one embodiment, the non-coding RNA edited by the method provided herein is selected from the group consisting of microRNA, small interfering RNA, transfer RNA, ribosomal RNA, trans-acting small interfering RNA, naturally occurring antisense small interfering RNA, heterochromatin small interfering RNA, and their precursors. In yet another embodiment, the gene edited by the method provided herein encodes miRNA. In a further embodiment, the gene edited by the method provided herein encodes precursor miRNA (pre-miRNA).

[0245] In one embodiment, the GA20 oxidase gene provided herein is encoded by mRNA encoding a protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs (inserting the protein sequence of GA20). In another embodiment, the brachytic2 gene provided herein is encoded by mRNA encoding a protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs (inserting the protein sequence of BR2).

[0246] In one embodiment, the unmodified alleles provided herein include a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity or complementarity with a sequence selected from the group consisting of sequence numbers (listing GA and BR2 sequences).

[0247] In another aspect, the non-coding RNAs edited by the methods provided herein are selected from the group consisting of microRNAs, small interfering RNAs, transfer RNAs, ribosomal RNAs, trans-acting small interfering RNAs, naturally occurring antisense small interfering RNAs, heterochromatin small interfering RNAs, and their precursors. [Examples]

[0248] Example 1. Generation of GA20 oxidase dominant allele via targeted genomic inversion We created two functional guide RNAs (gRNAs) for a CRISPR / RNA-guided nuclease system that target the flanking regions (left target site and right target site) of the GA20 oxidase_5 gene in the maize genome. See Figure 1, Panel A. Each of the two target sites is unique within the maize genome. The plant hormone gibberellins play important roles in several plant developmental processes, including germination, cell elongation, flowering, embryogenesis, and seed development. Specific biosynthetic enzymes (e.g., GA20 oxidase and GA3 oxidase) and catabolic enzymes (e.g., GA2 oxidase) in the GA pathway are important for influencing GA levels in plant tissues.

[0249] A transfer DNA (T-DNA) vector suitable for Agrobacterium transformation is used. The T-DNA construct contains several expression cassettes between the left-bound (LB) and right-bound (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to polynucleotides encoding the two gRNAs mentioned above.

[0250] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides a nuclease to each of two target sites in the GA20 oxidase_5 gene, and the nuclease produces a double-strand break at each target site.

[0251] In most cases, a region between target sites is deleted, and a non-homologous end repair mechanism junctions the flanking region. Less frequently, in some cases, insertion / deletion mutations are produced in the left target site, the right target site, or both. In yet another case, known as a "complete inversion," the entire targeted region is inverted. See Figure 1, Panel B. Transformation events, including complete inversions, are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transgenic embryos containing a complete inversion targeted at the 5' end of the GA20 oxidase_5 gene are selected and used to regenerate modified plants using standard techniques in this art.

[0252] While not bound by any particular scientific theory, the presence of a complete inversion in one allele of GA20 oxidase_5 generates a population of antisense mRNA under the control of the native GA20 oxidase_5 promoter. The inversion region of the edited GA20 oxidase_5 allele mRNA and the corresponding region in the unedited GA20 oxidase_5 allele mRNA are complementary and can form dsRNA. Therefore, even if the modified maize plant is heterozygous for the edited GA20 oxidase_5 allele, the edited allele can reduce the expression of the GA20 oxidase_5 gene in the modified maize plant.

[0253] RNA is extracted from modified maize plants identified as containing a complete inversion of the targeting region of the GA20 oxidase_5 gene. RNA is also extracted from control maize plants lacking the complete inversion. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing), downregulation of GA20 oxidase_5 occurs in the modified maize plants containing the complete inversion of the targeting region.

[0254] Example 2. Generation of BR2 dominant allele via targeted genomic inversion We will create two functional guide RNAs (gRNAs) for an RNA-guided nuclease system that target the flanking regions (left target site and right target site) of the BRACHYTIC2 (BR2) gene in the maize genome. Each of the two target sites is unique within the maize genome.

[0255] A transfer DNA (T-DNA) vector suitable for Agrobacterium tumefaciens transformation is used. The T-DNA construct contains several expression cassettes between the left-bound (LB) and right-bound (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to polynucleotides encoding the two gRNAs mentioned above.

[0256] Immature maize embryos are co-cultured with Agrobacterium tumefaciens containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides CRISPR endonucleases to each of two target sites in the BR2 gene, and the CRISPR endonucleases create double-strand breaks at each target site.

[0257] In most cases, a region between target sites is deleted, and a non-homologous end repair mechanism junctions the flanking region. Less frequently, in some cases, insertion / deletion mutations are produced at the left target site, the right target site, or both. In yet another case, known as a "complete inversion," the entire targeted region is inverted. Transformation events, including complete inversions, are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transgenic embryos containing a complete inversion targeted at the 5' end of the BR2 gene are selected and used to regenerate plants using standard techniques in this art.

[0258] While not bound by any particular scientific theory, the presence of a complete inversion in one allele of the BR2 gene generates a population of antisense mRNA under the control of the native BR2 promoter. The inverted region of the edited BR2 allele mRNA and the corresponding region in the unedited BR2 allele mRNA are complementary and can form dsRNA. Therefore, even if the maize plant is heterozygous for the edited BR2 allele, the edited allele can reduce the expression of both alleles of the BR2 gene in the maize plant, thus resulting in a short-stemmed phenotype.

[0259] Example 3. Generation of the dominant allele of GA20 oxidase via targeted genomic deletion under the control of two inverted promoters. The gene encoding GA20 oxidase_5 (also known as GA20ox5) is located on chromosome 8 of maize. It is adjacent to the maize gene GRMZM2G049269, which encodes S-adenosyl-L-methionine-dependent methyltransferase superfamily protein (hereinafter referred to as "SAMT"). SAMT is a member of a large duplicate gene family, and phenotypes associated with mutations in this gene have not been reported in maize or Arabidopsis. The SAMT gene is oriented in the opposite direction to the GA20 oxidase_5 gene (i.e., the SAMT gene is oriented to be read from 5' to 3', while the GA20 oxidase_5 gene is oriented to be read from 3' to 5' on the same DNA strand). See Figure 2, Panel A.

[0260] Two functional guide RNAs (gRNAs) for an RNA-guided nuclease system are created, targeting the genomic DNA region between the GA20 oxidase_5 gene and the SAMT gene. The first gRNA targets an area near the transcription start site of the SAMT gene, and the second gRNA targets a region near the transcription termination site of the GA20 oxidase_5 gene. Each of the two target sites is unique within the maize genome. A transfer DNA (T-DNA) vector suitable for Agrobacterium transformation is used. The T-DNA construct contains several expression cassettes between the left boundary (LB) sequence and the right boundary (RB) sequence. The first expression cassette contains a plant cell-operable promoter operably ligated to a polynucleotide encoding the RNA-guided nuclease. The second expression cassette contains a plant cell-operable promoter operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a plant cell-operable promoter operably ligated to the polynucleotide encoding the two gRNAs described above.

[0261] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides nucleases to two target sites within the genomic DNA region between the GA20 oxidase_5 gene and the SAMT gene, and the nucleases produce double-strand breaks at each target site.

[0262] In most cases, a region between target sites is deleted, and a non-homologous end repair mechanism junctions the flanking region. Transformation events, including complete deletions, are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing targeted deletions between the GA20 oxidase_5 gene and the SAMT gene are selected and used to regenerate modified plants using techniques standard in the art.

[0263] While not bound by any particular scientific theory, by removing genomic DNA between the GA20 oxidase_5 gene and the SAMT promoter, the native SAMT promoter can generate an antisense mRNA transcript of the GA20 oxidase_5 gene, and the native GA20 oxidase_5 promoter can generate a sense mRNA transcript of the GA20 oxidase_5 gene. The complementary sense and antisense mRNA transcripts of the GA20 oxidase_5 gene can form dsRNAs that can be processed by RNA silencing mechanisms specific to maize cells. See Figure 2, Panel B. The processed dsRNAs can then suppress the expression of both alleles of GA20 oxidase_5. Furthermore, since the mRNA encoding GA20 oxidase_3 is very similar to the sense GA20 oxidase_5 transcript, deletion between the SAMT promoter and the GA20 oxidase_5 gene is also expected to downregulate the expression of GA20 oxidase_3. Furthermore, it is assumed that the repression or silencing of the GA20 oxidase_5 gene may occur by other mechanisms provided herein (e.g., nonsense-mediated disruption) instead of, or in addition to, the repression of any RNAi or PTGS morphology.

[0264] RNA is extracted from modified maize plants identified as containing a targeted deletion between the SAMT promoter and the GA20 oxidase_5 gene. RNA is also extracted from control maize plants lacking the deletion. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing), it is confirmed that downregulation of GA20 oxidase_5 and / or GA20 oxidase_3 occurs in the modified maize plants containing the targeted region deletion.

[0265] Example 4. Generation of the dominant allele of BR2 via targeted genomic deletion under the control of two inverted promoters of the BR2 gene. The gene encoding BR2 is located on chromosome 1 of maize. It is adjacent to the maize gene GRMZM2G491632, which is expressed in the opposite direction to BR2. Two functional guide RNAs (gRNAs) are created for an RNA-guided nuclease system that target the genomic DNA region between the BR2 gene and the GRMZM2G491632 gene. The first gRNA targets an area near the end of exon 1 of the BR2 gene, and the second gRNA targets a region near the beginning of the coding sequence of exon 1 of the GRMZM2G491632 gene.

[0266] A transfer DNA (T-DNA) vector suitable for Agrobacterium transformation is used. The T-DNA construct contains several expression cassettes between the left-bound (LB) and right-bound (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to polynucleotides encoding the two gRNAs mentioned above.

[0267] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides endonucleases to two target sites within the genomic DNA region between the BR2 gene and the GRMZM2G491632 gene, and the endonucleases produce double-strand breaks at each target site.

[0268] In most cases, a region between target sites is deleted, and a non-homologous end repair mechanism junctions the flanking region. Transformation events containing the deletion are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing targeted deletions between the BR2 gene and the GRMZM2G491632 gene are selected and used to regenerate plants using standard techniques in the art.

[0269] While not bound by any particular scientific theory, by removing genomic DNA between the BR2 gene and the GRMZM2G491632 promoter, the native GRMZM2G491632 promoter can generate an antisense mRNA transcript of the BR2 gene, and the native BR2 promoter can generate a sense mRNA transcript of the BR2 gene. The complementary sense and antisense mRNA transcripts of the BR2 gene can form dsRNAs that can be processed by RNAi mechanisms specific to maize cells. The processed dsRNAs can repress the expression of both BR2 alleles, thus resulting in a short-stemmed phenotype.

[0270] Example 5. Creation of a dominant allele by inserting an element designed using genome editing technology into a native promoter. Genes containing root-specific promoters have been identified in the Arabidopsis thaliana genome. See Figure 3, Panel A. This promoter is used to drive the expression of GUS in the roots of plants. See Figure 3, Panel B. Design a functional gRNA to target the region upstream of the TATA box of the root-specific promoter (the "target site"). Use a transfer DNA (T-DNA) vector suitable for Agrobacterium transformation to introduce the gRNA into Arabidopsis. The T-DNA construct contains several expression cassettes between the left border (LB) sequence and the right border (RB) sequence. The first expression cassette contains a promoter operable in plant cells, operably linked to a polynucleotide encoding an RNA-guided nuclease. The second expression cassette contains a promoter operable in plant cells, operably linked to the CP4-EPSPS marker gene. The construct also includes an expression cassette containing a promoter operable in plant cells, operably linked to the polynucleotide encoding the gRNA described above. The second T-DNA construct contains a polynucleotide encoding a donor molecule that includes an element designed to be inserted at the target site between the LB and RB sequences. In one embodiment, the donor molecule includes a designed element flanked by homologous regions homologous to the sequences present on either side of the target site. The designed element allows a gene that was previously root-specific to be constitutively expressed in all tissues of the plant when inserted into the promoter region of the gene. In another embodiment, the donor molecule contains a designed sequence flanked by the target site targeted by the gRNA of T-DNA vector 1.

[0271] The floral dip method is used to transform Arabidopsis using the vector described above. See Clough and Bent, 1998, Plant J, 16:735-743, incorporated herein by reference. When the incorporated polynucleotide is expressed, the gRNA guides a nuclease to the target site, creating a double-strand break at the target site. In the case of a donor molecule containing a designed sequence that is flanked by homologous arms, a homologous repair mechanism unique to Arabidopsis cells inserts the designed element into the double-strand break site. In the case of a donor molecule containing a designed sequence that is flanked by a gRNA target site, the gRNA guides a nuclease to create a double-strand break in a second T-DNA, thereby releasing the designed sequence, which may then be incorporated into the genomic target site via the NHEJ (non-homologous end joining) repair mechanism. In some insertion events, the promoter inserts in the desired orientation. While not bound by any particular theory, the presence of a designed element upstream of the TATA box induces constitutive expression of the gene throughout the modified plant, thereby creating a dominant allele of the gene.

[0272] Using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing), transformation events involving targeted insertions of elements designed in a desired orientation are identified. Transformed Arabidopsis plants containing the designed elements at the target site are selected and further examined.

[0273] Plants identified as containing elements designed upstream of the TATA box (see Figure 3, Panel D) are examined for GUS expression using standard techniques in the art. Plants containing the designed elements exhibit GUS expression throughout the plant. See Figure 3, Panel C. Further, RNA is extracted from various tissues (e.g., roots, stems, leaves, inflorescences) of modified Arabidopsis plants identified as containing the designed elements. RNA is also extracted from control Arabidopsis plants lacking the designed elements. Suitable methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing) are used to confirm that GUS is expressed more widely and / or more strongly in the modified Arabidopsis plants.

[0274] Example 6. Generation of dominant alleles by inserting tissue-specific silencing elements using genome editing techniques. Using standard techniques in the art, Arabidopsis thaliana plants containing the GUS transgene under the control of a functional promoter in the leaf, vascular, and root tissues are created. For an overview of the concepts provided in this embodiment, see Figure 4 and Figure 5, panels A and B. A functional gRNA is designed to target the downstream region of the GUS gene ("target site"). The gRNA is introduced into Arabidopsis using a transfer DNA (T-DNA) vector suitable for use in Agrobacterium transformation. The T-DNA construct contains several expression cassettes between the left-boundary (LB) and right-boundary (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to a polynucleotide encoding the aforementioned gRNA. The second T-DNA construct includes a donor molecule containing a leaf-specific promoter, such as the COOLAIR promoter (see Chen and Penfield, Science, 2018, 360:6392), between the LB and RB sequences. In one embodiment, the donor molecule includes an antisense-oriented promoter that is flanked by a homologous region homologous to a sequence located on either side of the target site. The antisense-oriented leaf-specific promoter enables the expression of GUS antisense mRNA in the tissue in which the promoter is expressed (e.g., leaf tissue). While not bound by any particular theory, the antisense RNA transcript of the GUS gene causes GUS silencing in leaf tissue but not in root tissue. In another embodiment, the donor molecule includes a promoter sequence that is flanked by a target site targeted by the gRNA of T-DNA vector 1.

[0275] The floral dip method is used to transform Arabidopsis using the vector described above. See Clough and Bent, 1998, Plant J, 16:735-743, incorporated herein by reference. Upon polynucleotide expression, the gRNA guides a nuclease to the target site, creating a double-strand break at that site. In the case of a donor molecule containing an antisense-oriented promoter flanked by a homologous arm, the homologous repair mechanism unique to Arabidopsis cells inserts a leaf-specific promoter at the site of the double-strand break downstream of the GUS gene, thereby orienting the promoter antisense relative to the GUS gene. In the case of a donor molecule containing a promoter flanked by a gRNA target site, the gRNA guides a nuclease to create a double-strand break within a second T-DNA, thereby releasing the promoter sequence, which can then be incorporated into the genomic target site via the NHEJ (non-homologous end joining) repair mechanism. In some insertion events, the promoter is inserted in an antisense orientation. While not bound by any particular theory, the presence of an antisense leaf-specific promoter induces a reduction in GUS expression throughout the plant's leaf tissue, thereby creating a dominant allele of the gene.

[0276] Using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing), transformation events involving targeted promoter insertions in a desired orientation are identified. Transformed Arabidopsis plants containing leaf-specific promoters at the target site are selected and further examined.

[0277] Plants identified as containing a leaf-specific promoter downstream of the GUS gene in an antisense orientation relative to the GUS gene (see Figure 5, Panel D) are examined for GUS expression using standard techniques in the art. Plants containing a leaf-specific promoter in an antisense orientation relative to the GUS gene exhibit GUS expression only in root tissue. See Figure 5, Panel C. Furthermore, RNA is extracted from various tissues (e.g., root, stem, leaves, inflorescence) of the modified Arabidopsis plants identified as containing a leaf-specific promoter. RNA is also extracted from control Arabidopsis plants lacking a leaf-specific promoter in an antisense orientation relative to the GUS gene. GUS expression is confirmed to be reduced in leaf tissue using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing).

[0278] Example 7. Creation of a dominant GA20 oxidase_5 allele by inserting a tissue-specific repressive element using genome editing technology. A functional gRNA is designed to target the downstream region of the 3'-UTR of the GA20 oxidase_5 gene ("target site"). The gRNA is introduced into maize cells using a transfer DNA (T-DNA) vector suitable for Agrobacterium transformation. The T-DNA construct contains several expression cassettes between the left-boundary (LB) sequence and the right-boundary (RB) sequence. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to a polynucleotide encoding the aforementioned gRNA. The second T-DNA construct contains a donor molecule containing an RTBV promoter between the LB sequence and the RB sequence. In one embodiment, the donor molecule includes an antisense-oriented RTBV promoter that is flanked by a homologous region homologous to a sequence located on either side of the target site. In another embodiment, the donor molecule includes a promoter sequence that is flanked by a target site targeted by the gRNA of T-DNA vector 1. The antisense-oriented RTBV promoter enables the expression of GA20 oxidase_5 antisense mRNA in tissues where RTBV is expressed (e.g., stem and vascular tissue). Although not bound by any particular theory, antisense RNA transcripts of the GA20 oxidase_5 gene cause silencing of both GA20 oxidase_5 and GA20 oxidase_3 in stem and vascular tissue.

[0279] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. Upon polynucleotide expression, the gRNA guides a nuclease to a target site, creating a double-strand break at that site. In the case of a donor molecule containing an antisense-oriented RTBV promoter that is flanked by a homologous arm, a homologous repair mechanism specific to maize cells inserts the antisense RTBV promoter into a target site downstream of the 3' end of the GA20 oxidase_5 gene. In the case of a donor molecule containing a promoter that is flanked by a gRNA target site, the gRNA guides a nuclease to create a double-strand break within a second T-DNA, thereby releasing the promoter sequence, which can then be incorporated into a genomic target site via the NHEJ (non-homologous end joining) repair mechanism. In some insertion events, the promoter is inserted in an antisense orientation.

[0280] Transformation events involving targeted insertions of antisense RTBV promoters are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing the antisense RTBV promoter at the target site are selected and used to regenerate modified plants using art-standard techniques.

[0281] RNA is extracted from various tissues (e.g., roots, stems, leaves, inflorescences) of modified maize plants identified as containing an antisense RTBV promoter. RNA is also extracted from control maize plants lacking the antisense RTBV promoter at the target site. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing), the expression of GA20 oxidase 5 and / or GA20 oxidase 3 is reduced in the stem and vascular tissues of the modified maize plants containing the antisense RTBV promoter compared to the control maize plants.

[0282] Example 8. Creation of dominant alleles by manipulating cleavage-type proteins using genome editing technology. A) Manipulation of GA20 oxidase cleavage proteins: Dominant alleles can be created by targeted editing of genes that result in cleavage proteins or nonsense mutations in proteins. See Figure 6. The maize GA20 oxidase_5 and GA20 oxidase_3 genes are highly similar in sequence and structure. Both genes contain three exons. Design gRNAs to introduce edits into the exons of both genes.

[0283] gRNA is introduced into maize cells using a transfer DNA (T-DNA) vector suitable for Agrobacterium transformation. The T-DNA construct contains several expression cassettes between the left-boundary (LB) and right-boundary (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to the polynucleotide encoding the aforementioned gRNA.

[0284] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides a nuclease to a target site, creating a double-strand break at that site. While not bound by any particular theory, cell-specific non-homologous end repair mechanisms frequently repair such breaks incompletely, which can lead to the insertion or deletion of one or more nucleotides. Such insertions or deletions into exons can result in immature stop codons (which produce cleavage proteins) or nonsense mutations. Immature stop codons have the ability to produce dominant alleles of GA20 oxidase_5 and GA20 oxidase_3.

[0285] Using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing), transformation events involving insertions or deletions targeted to the GA20 oxidase_5 and / or GA20 oxidase_3 genes are identified. Transformed embryos containing insertions or deletions at the target site, which can introduce immature stop codons or nonsense mutations resulting in cleavage proteins, are selected and used to regenerate modified plants using techniques standard in the art.

[0286] Extract proteins from identified modified maize plants containing the identified insertions / deletions. Confirm that cleavage proteins or nonsense mutations have been introduced into the GA20 oxidase_5 and / or GA20 oxidase_3 genes using preferred methods known in the art (e.g., Western blotting, HPLC, LC / MS, ELISA, immunoprecipitation). Perform additional experiments to confirm the reduction of gibberellates in stem and / or vascular tissue as described in Bensen et al., Plant Physiol. 1990, 94:77-84, which is incorporated herein by reference in whole.

[0287] B) Manipulation of Brachytic 2 (Br2) cleavage proteins: Design gRNA to introduce editing into the exons of the maize-derived Brachytic 2 gene.

[0288] gRNA is introduced into maize cells using a transfer DNA (T-DNA) vector suitable for Agrobacterium transformation. The T-DNA construct contains several expression cassettes between the left-boundary (LB) and right-boundary (RB) sequences. The first expression cassette contains a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette contains a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also contains an expression cassette containing a promoter operable in plant cells, operably ligated to the polynucleotide encoding the aforementioned gRNA.

[0289] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides a nuclease to a target site, where the nuclease creates a double-strand break. While not bound by any particular theory, cell-specific non-homologous end repair mechanisms frequently repair such breaks incompletely, which can lead to the insertion or deletion of one or more nucleotides. Such insertions or deletions into exons can result in immature stop codons (which produce break-type proteins) or nonsense mutations. Immature stop codons have the ability to produce the dominant allele of Brachytic 2 (Br2).

[0290] Using suitable methods known in the art (e.g., PCR, DNA hybridization (Southern) blot, sequencing), identify transformation events containing insertions or deletions targeted to the Br2 gene. Select transformed embryos containing insertions or deletions in the target site that can introduce premature stop codons (resulting in truncated proteins) or nonsense mutations, and use them to regenerate modified plants using standard techniques in the art.

[0291] Extract proteins from the identified modified maize plants containing the identified insertion / deletion. Use suitable methods known in the art (e.g., Western blot, HPLC, LC / MS, ELISA, immunoprecipitation) to confirm that the truncated Br2 protein is being produced.

[0292] Example 9. Generation of Inverted Repeats in a Target Gene Using targeted editing techniques, it is possible to convert a genomic locus such that when the edited locus is transcribed into RNA, it becomes a locus capable of generating an RNAi-inducing hairpin. Refer to Figure 7. In cells that are heterozygous at the locus of interest (e.g., where two polymorphic alleles are present), one or more nucleases are used to generate two double-strand breaks (e.g., a first double-strand break and a second double-strand break) in the first allele and one double-strand break (e.g., a third double-strand break) in the second allele. When the nuclease cleaves the first and second alleles, a portion of the first allele that is flanked by the first and second double-strand breaks is released from the genomic DNA. One result is that the orientation of the released portion of the first allele is inverted and incorporated into the third double-strand break in the second allele, thereby creating an edited locus capable of generating an RNAi-inducing hairpin when the edited locus is transcribed.

[0293] We create first and second functional guide RNAs (gRNAs) for an RNA-guided nuclease system. The first and second gRNAs are complementary to the first and second target sites, respectively, which flank a portion of the first allele of the GA20 oxidase_5 gene in the maize genome. The first gRNA is also complementary to the second allele of the GA20 oxidase_5 gene at a third target site (homologous to the first target site), but the second gRNA is not complementary to the second allele due to polymorphism between the first and second GA20 oxidase_5 alleles at the second target site.

[0294] A transfer DNA (T-DNA) vector suitable for use in Agrobacterium transformation is constructed. The T-DNA construct includes several expression cassettes between the left-bound (LB) sequence and the right-bound (RB) sequence. The first expression cassette includes a promoter operable in plant cells, operably ligated to a polynucleotide encoding an RNA guide nuclease. The second expression cassette includes a promoter operable in plant cells, operably ligated to the CP4-EPSPS marker gene. The construct also includes an expression cassette containing a promoter operable in plant cells, operably ligated to the polynucleotides encoding the first and second gRNAs described above.

[0295] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. When polynucleotides are expressed, the gRNA guides nucleases to each of the three target sites within the GA20 oxidase_5 allele, and the nucleases produce double-strand breaks at each target site.

[0296] In most cases, a deletion occurs in the region between the first and second target sites in the first GA20 oxidase_5 allele, and the non-homologous end repair mechanism ligates the flanking region. Less frequently, in some cases, insertion / deletion mutations are produced at the first target site, the second target site, or both. In yet another case, the entire targeting region of the first allele is incorporated in reverse to a double-strand break at the third target site. See Figure 7, Panel D. Transformation events containing inversions in the second GA20 oxidase_5 allele are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing inversions in the second GA20 oxidase_5 allele are selected and used to regenerate modified plants using art-standard techniques.

[0297] While not bound by any particular scientific theory, the presence of an inversion in one allele of GA20 oxidase_5 creates a population of RNA transcripts capable of forming a hairpin structure. Such hairpins induce RNAi mechanisms within the cell, leading to dominant downregulation of GA20 oxidase_5 RNA transcripts (e.g., both edited and unedited alleles are downregulated).

[0298] RNA is extracted from modified maize plants identified as containing an inversion in the second allele capable of producing hairpin RNA transcripts. RNA is also extracted from control maize plants lacking the edited GA20 oxidase_5 allele. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing), it is confirmed that downregulation of GA20 oxidase_5 occurs in modified maize plants containing an inversion in the second allele capable of producing hairpin RNA transcripts. Furthermore, because GA20 oxidase_5 and GA20 oxidase_3 are sequence-similar, the inversion in the second allele of GA20 oxidase_5 also leads to downregulation of GA20 oxidase_3 RNA transcripts.

[0299] Example 10. Insertion of a miRNA target site into a desired genomic locus. Using targeted editing techniques, donor molecules can be inserted into target sites in genomic loci. See Figure 8. When a donor molecule containing a non-coding RNA target site is inserted into the 5'-UTR, exon, intron, or 3'-UTR of the gene of interest, RNA transcription or protein translation of the gene of interest can be repressed by complementary non-coding RNA. If the gene of interest is targeted by non-coding RNA (e.g., miRNA or siRNA), the cleaved mRNA of the gene of interest can generate secondary siRNA, which can further repress the transcription or translation of the gene of interest. Since secondary siRNA is complementary to the allele with or without the insertion of the non-coding RNA target site, such secondary repression can act in a dominant manner.

[0300] A functional guide RNA (gRNA) complementary to the target site in the 3'-UTR of the GA20 oxidase_5 gene is created. A transfer DNA (T-DNA) vector suitable for use in Agrobacterium transformation is constructed. The T-DNA construct contains a plant cell-operable promoter operably ligated between the left boundary (LB) sequence and the right boundary (RB) sequence to a) an RNA guide / nuclease, b) a CP4-EPSPS marker gene, c) the aforementioned gRNA, and d) a polynucleotide encoding a donor molecule. The donor molecule contains a 21-nucleotide sequence homologous to miR166, as well as first and second homologous regions homologous to the 3'-UTR of the GA20 oxidase_5 gene on either side of the target site.

[0301] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. Upon polynucleotide expression, the gRNA guides a nuclease to a target site in the 3'-UTR of GA20 oxidase_5, where the nuclease produces a double-strand break. Subsequently, the homologous recombination repair mechanism inserts a donor molecule into the target site, thereby integrating the miR166 target site into the 3'-UTR of the GA20 oxidase_5 gene.

[0302] Using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing), transformation events involving the insertion of the miR166 target site into the 3'-UTR of the GA20 oxidase_5 gene are identified. Transformed embryos containing the insertion of the miR166 target site into the 3'-UTR of the GA20 oxidase_5 gene are selected and used to regenerate modified plants using art-standard techniques.

[0303] While not bound by any particular scientific theory, the presence of a miRNA binding site on GA20 oxidase 5 creates a population of secondary siRNA transcripts capable of repressing GA20 oxidase 5 RNA transcription in a dominant-negative manner.

[0304] RNA is extracted from modified maize plants identified as containing a miR166 target site insertion in the 3'-UTR of the GA20 oxidase_5 gene. RNA is also extracted from control maize plants lacking the edited GA20 oxidase_5 gene. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, reverse transcriptase PCR, RNA sequencing), downregulation of GA20 oxidase_5 occurs in the modified maize plants. Furthermore, because GA20 oxidase_5 and GA20 oxidase_3 are sequence-similar, the miRNA target site in the GA20 oxidase_5 gene can also induce downregulation of the GA20 oxidase_3 RNA transcript.

[0305] Example 11. Generation of dominant-negative alleles by creating cleavage-type proteins. Targeted editing of genes resulting in cleavage proteins (e.g., nonsense mutations) can create dominant-negative alleles. In one embodiment, the targeted gene encodes a protein having a protein-protein interaction domain. See Figure 6. Examples of dominant cleavage proteins are known in various plant species. For example, dominant mutant phenotypes caused by cleavage proteins are known in FAZ1 of rice, and in AGAMOUS and SOC1 of Arabidopsis thaliana. The peptide CLAVATA3 (CLV3) is processed into a signal peptide (CLE), to which the receptor-like kinases CLV1 and CORYNE (CRN) and the receptor-like protein CLV2 bind to regulate the expression of WUSCHEL (WUS) at the growing point. Maize plants containing the CLV2 mutant allele often exhibit ears with an increased number of grain rows. While not limited to a specific theory, mutations in CLV2 may increase WUS expression, which in turn increases the size of the growing point and consequently increases the number of grain rows on the ear. CLV2 contains an extracellular domain and a transmembrane domain and forms a complex with CRN. The cleaved CLV2 protein can function in a dominant manner to increase the size of the maize growth point.

[0306] The gRNA is designed to introduce a stop codon into the extracellular domain of maize CLV2. The gRNA is introduced into maize cells using a transfer DNA (T-DNA) vector suitable for Agrobacterium transformation. The T-DNA construct includes a plant cell-operable promoter operably ligated between the left-boundary (LB) sequence and the right-boundary (RB) sequence to a) an RNA guide nuclease, b) a CP4-EPSPS marker gene, and c) a polynucleotide encoding the aforementioned gRNA.

[0307] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, the gRNA guides a nuclease to a target site, creating a double-strand break at that site. Although not bound by either theory, cell-specific non-homologous end repair mechanisms frequently repair such breaks incompletely, which can lead to the insertion or deletion of one or more nucleotides. Such insertions or deletions into exons can result in immature stop codons (which produce cleavage proteins) or nonsense mutations. Such mutations can generate a dominant-negative allele of CLV2.

[0308] Using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing), transformation events involving insertions or deletions targeted to the CLV2 gene are identified. Transformed embryos containing insertions or deletions at target sites capable of causing cleavage proteins are selected and used to regenerate modified plants using techniques standard in the art.

[0309] Proteins are extracted from modified maize plants that have been identified as containing the identified insertion / deletion. Confirmation that the nonsense mutation has been introduced into the CLV2 gene is made using preferred methods known in the art (e.g., Western blotting, HPLC, LC / MS, ELISA, immunoprecipitation). Additional phenotypic screening of the grain rows of the panicle is performed for increased meristem size. Light microscopy of meristem sections from modified and control plants is also performed to quantify the increase in meristem size.

[0310] Example 12. Prevention of target gene cleavage using a modified PPR protein. Members of the pentatricopeptide repeat (PPR) gene family are common in plant genomes. Many PPR proteins can bind to RNA molecules in a sequence-specific manner. A PPR protein contains 2 to 30 PPR motifs, each of which aligns with a single nucleotide within the RNA molecule. Within a PPR motif, the presence of two or three specific amino acids confers nucleotide specificity. For example, but are not limited to, when threonine is at position 6 and asparagine is at position 1', the PPR motif binds to an adenine nucleotide; when threonine is at position 6 and aspartic acid is at position 1', the PPR motif binds to a guanine nucleotide; when asparagine is at position 6 and aspartic acid is at position 1', the PPR motif binds to a uracil (or thymine) nucleotide; and when asparagine is at position 6 and asparagine or serine is at position 1', the PPR motif binds to a cytosine nucleotide.

[0311] While not limited to these, engineered PPR proteins can be generated by at least two construction strategies. In the first strategy, the PPR protein is constructed by treating each PPR motif as a separate block, so that the PPR protein is constructed by arranging multiple desired motifs. Thus, the resulting engineered PPR protein can bind to a target RNA molecule. However, this strategy does not always work because each PPR motif contains an internal scaffold between the 1' and 6' positions, and this internal motif scaffold is not shared between different PPR proteins. The second strategy utilizes an existing internal motif scaffold. In the second strategy, site-directed mutagenesis at the 1' and 6' positions is used to edit an existing PPR protein to make it specific to a new target RNA molecule.

[0312] The engineered PPR protein, containing a PPR motif, is engineered so that the PPR protein can specifically bind to nucleotides at the miRNA target site of a suitable gene and target the cytoplasm. The nucleic acid sequence encoding the engineered PPR protein is inserted into a transfer DNA (T-DNA) vector suitable for use in Agrobacterium transformation. The nucleic acid sequence encoding the engineered PPR protein is operably ligated to the promoter or constitutive promoter of the target gene to ensure double expression of the engineered PPR protein and the target gene mRNA. The nucleic acid molecules encoding the engineered PPR protein and the operably ligated promoter are located between the left boundary (LB) and right boundary (RB) sequences in the T-DNA construct.

[0313] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, a modified PPR protein is expressed and binds to a complementary target mRNA.

[0314] Transformation events involving the insertion of nucleic acid molecules encoding the engineered PPR protein are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing the insertion of nucleic acid molecules encoding the engineered PPR protein are selected and used to regenerate modified plants using techniques standard in the art.

[0315] While not bound by any particular scientific theory, the manipulated PPR protein binds to the target gene mRNA, preventing the microRNA from repressing the target gene.

[0316] RNA and proteins are extracted from modified maize plants identified as containing the manipulated PPR protein. RNA and proteins are also extracted from control maize plants lacking the manipulated PPR protein. Using preferred methods known in the art (e.g., quantitative reverse transcriptase PCR, RNA sequencing, Western blotting, HPLC, LC / MS, ELISA, immunoprecipitation), it is confirmed that the expression of the target gene is increased in the modified maize plants compared to the unmodified control maize plants.

[0317] Example 13. Cleavage of target gene transcripts using a modified PPR protein coupled to a nuclease. The engineered PPR protein, containing a NYN nuclease domain and a PPR motif, is engineered as described in Example 12 so that the PPR protein can specifically bind to a particular nucleotide of the target gene and so that the PPR protein targets the nucleus. The nucleic acid sequence encoding the engineered PPR protein is inserted into a transfer DNA (T-DNA) vector suitable for use in Agrobacterium transformation. The nucleic acid sequence encoding the engineered PPR protein is operably ligated to the promoter or constitutive promoter of the target gene to ensure double expression of the engineered PPR protein and the target gene mRNA. The nucleic acid molecules encoding the engineered PPR protein and the operably ligated promoter are located between the left boundary (LB) sequence and the right boundary (RB) sequence in the T-DNA construct.

[0318] Immature maize embryos are co-cultured with Agrobacterium containing a T-DNA vector for 3 days. A polynucleotide between the LB and RB sequences is incorporated into the nuclear genome of the immature maize embryos. When the incorporated polynucleotide is expressed, a modified PPR protein is expressed and binds to a complementary target mRNA.

[0319] Transformation events involving the insertion of nucleic acid molecules encoding the engineered PPR protein are identified using preferred methods known in the art (e.g., PCR, DNA hybridization (Southern) blotting, sequencing). Transformed embryos containing the insertion of nucleic acid molecules encoding the engineered PPR protein are sel...

Claims

1. A method for generating a dominant-negative allele of a gene in a cell, comprising deleting a portion of a chromosome, wherein the chromosome comprises a first gene region including a first promoter and a first coding region, and a second gene region including a second promoter and a second coding region, the first gene region and the second gene region being separated by an intervening region, the first promoter and the second promoter being positioned in opposite directions, and the deletion being performed using a targeted editing technique, wherein an antisense RNA transcript of the first coding region is generated by the second promoter due to the deletion of the second coding region and the intervening region.

2. a) Identifying a chromosomal region comprising a first gene region including a first promoter and a first coding region, and a second gene region including a second promoter and a second coding region, wherein the first coding region and the second coding region are separated by an intervening region, and the first promoter and the second promoter are positioned in opposite directions. b) Inducing a first double-strand break and a second double-strand break that flank in the target region including the second coding region and the intervening region, c) Identifying one or more cells containing the deletion of the target region of the chromosome, and d) Selecting one or more cells that include the deletion in the targeted region of the chromosome. A method including, A method wherein an antisense RNA transcript of the first coding region is generated by the second promoter due to deletion of the second coding region and the intervening region.

3. A method for reducing gene expression in cells, a) Identifying a chromosomal region comprising a first gene region including a first promoter and a first coding region, and a second gene region including a second promoter and a second coding region, wherein the first coding region and the second coding region are separated by an intervening region, and the first promoter and the second promoter are positioned in opposite directions. b) Inducing a first double-strand break and a second double-strand break to flank a targeted region using targeted editing techniques, wherein the targeted region includes the second coding region and the intervening region, and c) Identifying one or more cells containing the deletion of the targeting region. Includes, A method wherein the second promoter generates at least one antisense RNA of the first coding region, and the expression of the first coding region is reduced compared to a control cell that does not contain the deletion of the targeted region.

4. a) Identifying a chromosomal region comprising a first gene region including a first promoter and a first coding region, and a second gene region including a second promoter and a second coding region, wherein the first coding region and the second coding region are separated by an intervening region, and the first promoter and the second promoter are positioned in opposite directions. b) Providing one or more cells with at least one RNA guide nuclease, or one or more vectors encoding at least one RNA guide nuclease, The at least one RNA guide nuclease can bind to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 consecutive nucleotides of a first target site and a second target site that flank to a targeting region of a chromosome, wherein the targeting region includes the second coding region and the intervening region. The present invention provides an RNA guide nuclease that generates double-strand breaks at the first and second target sites of the chromosome. c) Identifying one or more cells containing the deletion of the targeting region, and d) Selecting one or more cells that include the deletion in the targeting region. A method that includes this.

5. A modified plant cell comprising a non-transposon-mediated genomic deletion of a portion of a chromosome, wherein the chromosome comprises a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first gene region and the second gene region are separated by an intervening region, the first promoter and the second promoter are positioned in opposite directions, the deletion of the second coding region and the intervening region results in the production of an RNA transcript by the second promoter, and the RNA transcript comprises a sequence complementary to the native transcript sequence of the first coding region or a portion thereof.

6. A modified chromosome comprising a non-transposon-mediated deletion of a portion of a chromosome, wherein the chromosome comprises a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first gene region and the second gene region are separated by an intervening region, the first promoter and the second promoter are positioned in opposite directions, the deletion of the second coding region and the intervening region results in the production of an RNA transcript by the second promoter, and the RNA transcript comprises a sequence complementary to the native transcript sequence of the first coding region or a portion thereof.

7. A modified cell comprising the modified chromosome described in claim 6.

8. A modified plant or modified plant tissue regenerated from the modified plant cells described in claim 5.

9. A product comprising the modified chromosome described in claim 6.

10. A product comprising the modified cells described in claim 7.

11. A modified plant or part thereof comprising a non-transposon-mediated genomic deletion of a portion of a chromosome, wherein the chromosome comprises a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first gene region and the second gene region are separated by an intervening region, the first promoter and the second promoter are positioned in opposite directions, and the deletion of the second coding region and the intervening region results in the production of an RNA transcript having a sequence complementary to the native transcript sequence of the first coding region or a portion thereof.

12. A product comprising the modified plant or a part thereof as described in claim 11.

13. A modified cell comprising a non-transposon-mediated genomic deletion of a portion of a chromosome, wherein the chromosome comprises a first gene region comprising a first promoter and a first coding region, and a second gene region comprising a second promoter and a second coding region, wherein the first gene region and the second gene region are separated by an intervening region, the first promoter and the second promoter are positioned in opposite directions, the deletion of the second coding region and the intervening region results in the production of an RNA transcript by the second promoter, and the RNA transcript comprises a sequence complementary to the native transcript sequence of the first coding region.

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