NZ711145B2 - Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair - Google Patents

Abstract

The invention provides to improved methods for the modification of genes in plant cells, and plants and seeds derived therefrom. More specifically, the invention relates to the increased efficiency of targeted gene mutation by combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms. In particular, the invention provides a method for introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target DNA sequence in a plant cell, comprising delivery of a GRON and a site-specific nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a TALEN and a CRISPR complex into a plant cell. The GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to attract the cell's gene repair system to the site where the mismatched base-pair(s) is created, and is degraded after designated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutation into the target DNA sequence and the plant cell is non-transgenic following the introduction. The GRON comprises one or more alterations from conventional RNA and DNA nucleotides at the 5' or 3' end. vailability of components of the target cell gene repair mechanisms. In particular, the invention provides a method for introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target DNA sequence in a plant cell, comprising delivery of a GRON and a site-specific nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a TALEN and a CRISPR complex into a plant cell. The GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to attract the cell's gene repair system to the site where the mismatched base-pair(s) is created, and is degraded after designated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutation into the target DNA sequence and the plant cell is non-transgenic following the introduction. The GRON comprises one or more alterations from conventional RNA and DNA nucleotides at the 5' or 3' end.

Description

(12) Granted patent specificaon (19) NZ (11) 711145 (13) B2 (47) Publicaon date: 2021.12.24 (54) METHODS AND COMPOSITIONS FOR INCREASING EFFICIENCY OF TARGETED GENE MODIFICATION USING OLIGONUCLEOTIDE-MEDIATED GENE REPAIR (51) Internaonal Patent Classificaon(s): A01H 1/00 A01H 5/00 (22) Filing date: (73) Owner(s): 2014.03.14 CIBUS US LLC CIBUS EUROPE B.V. (23) te specificaon filing date: 2014.03.14 (74) Contact: Wrays Pty Ltd (30) Internaonal Priority Data: US 61/801,333 2013.03.15 (72) Inventor(s): SCHOPKE, Christian (86) Internaonal Applicaon No.: SAUER, Noel, Joy PEARCE, James SEGAMI, Rosa, E. (87) Internaonal Publicaon number: MOZORUK, Jerry 4/144951 GOCAL, y, F.w.

BEETHAM, Peter, R. (57) Abstract: The on provides to improved methods for the aon of genes in plant cells, and plants and seeds derived therefrom. More specifically, the invenon relates to the increased efficiency of targeted gene mutaon by combining gene repair oligonucleodes with approaches that e the availability of components of the target cell gene repair isms. In parcular, the invenon provides a method for introducing a gene repair oligonucleobase (GRON)- mediated n into a target DNA sequence in a plant cell, comprising delivery of a GRON and a site-specific nuclease selected from the group consisng of a zinc finger nuclease (ZFN), a TALEN and a CRISPR complex into a plant cell. The GRON hybridizes at the target DNA sequence to create a mismatched base-pair(s), which acts as a signal to aract the cell's gene repair system to the site where the ched base-pair(s) is created, and is degraded aer designated nucleode(s) NZ 711145 B2 within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutaon into the target DNA sequence and the plant cell is non-transgenic ing the introducon. The GRON comprises one or more alteraons from convenonal RNA and DNA nucleodes at the 5' or 3' end.

S AND COMPOSITIONS FOR INCREASING EFFICIENCY OF TARGETED GENE MODIFICATION USING OLIGONUCLEOTIDE-MEDIATED GENE REPAIR The present application claims ty to US Provisional Patent Application 61/801,333 filed March 15, 2013, which is hereby incorporated by nce.

FIELD OF THE INVENTION This invention generally s to novel methods to improve the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences.

Additionally, this invention relates to target DNA that has been modified, mutated or marked by the approaches disclosed herein. The invention also relates to cells, tissue, and organisms which have been modified by the invention's methods.

BACKGROUND OF THE INVENTION The following discussion of the background of the invention is merely ed to aid the reader in understanding the invention and is not ed to describe or constitute prior art to the t invention.

The modification of genomic DNA is central to advances in biotechnology, in general, and biotechnologically based medical advances, in particular. Efficient methods for site— directed genomic ations are desirable for research and possibly for gene therapy applications. One approach utilizes triplex~forming oligonucleotides (TFO) which bind as third strands to duplex DNA in a ce-specific manner, to mediate directed mutagenesis.

Such TFO can act either by delivering a tethered mutagen, such as psoralen or chlorambucil (Havre et al., Proc Nat’l Acad Sci, USA. 90:7879—7883, 1993; Havre et al., J Virol 6717323~ 7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995; Takasugi et al., Proc Nat'l Acad Sci, USA. 88:5602—5606, 1991; Belousov et al., Nucleic Acids Res 25:3440—3444, 1997), or by binding with ent affinity to provoke error—prone repair (Wang et al., Science 271 :802—805, 1996).

Another strategy for genomic modification involves the induction of homologous recombination between an exogenous DNA fi‘agment and the targeted gene. This approach has been used sfully to target and disrupt selected genes in mammalian cells and has enabled the production of transgenic mice carrying specific gene knockouts (Capeechi et al., Science 244: 1288—1292, 1989; US. Pat. No. 191 to Wagner). This approach, however, relies on the transfer of selectable markers to allow 2014/029566 of homologous to non— ion of the desired recombinants. Without selection, the ratio is low, homologous integration of transfected DNA in typical gene er experiments W. H. Freeman and usually in the range of 121000 or less (Sedivy et al., Gene Targeting, Co., New York, 1992). This low efficiency of homologous integration limits the utility The frequency of homologous gene transfer for experimental use or gene y. ation and recombination can be enhanced by damage to the target site from UV well as by siteselected carcinogens (Wang et al., Mol Cell Biol 8:196—202, 1988) as and Co., New specific endonucleases (Sedivy et a1, Gene ing, W. H. Freeman et al., York, 1992; Rouet et al., Proc Nat’l Acad Sci, USA. 91:6064-6068, 1994; Segal Proc Nat’l Acad Sci, USA. 92:806—810, 1995). In addition, DNA damage induced by and between triplex-directed psoralen photoadducts can stimulate ination within extrachromosomal vectors (Segal et al., Proc Nat’l Acad Sci, USA. 92:806—810, 1995; Glazer).

Faruqi et al., Mol Cell Biol 16:6820—6828, 1996; US. Pat. No. 426 to Other work has helped to define parameters that influence recombination than their mammalian cells. In general, linear donor fragments are more recombinogenic Recombination is circular counterparts (Folger eta1., Mol Cell Biol 2: 1372—1387, 1982). both the donor and also influenced by the length of uninterrupted homology between be ineffective ates for recombination target sites, with short fragments appearing to (Rubnitz et al., Mol Cell Biol 4:2253~2258, 1984). Nonetheless, several recent efforts for gene have focused on the use of short fragments of DNA or DNA/RNA hybrids correction. (Kunzelmann et al., Gene Ther 32859—867, 1996). deliver a The sequence—specific binding properties of TFO have been used to series of different molecules to target sites in DNA. For example, a diagnostic method DNA cleaving for examining triplex interactions utilized TFO coupled to Fe—EDTA, a Others have linked biologically active agent (Moser et al., Science 238:645-650, 1987). and ococcal nuclease to TFO and demonstrated s like micrococcal se site-specific cleavage of DNA (Pei et al., Proc Nat’l Acad Sci USA. 8798589862, 1990; Landgraf et al., Biochemistry 33:10607—10615, 1994). Furthermore, site-directed either psoralen DNA damage and mutagenesis can be achieved using TFO conjugated to et al., Proc (Havre et al., Proc Nat’l Acad Sci USA. 9—7883, 1993; Takasurgi et al., Nucleic Nat’l Acad Sci USA. 88:5602—5606, 1991) or alkylating agents (Belousov Acids Res 25:3440-3444, 1997; Posvic et al., J Am Chem Soc 112:9428-9430, 1990). 2014/029566 bes methods for mutating a WIPO Patent Application WO/2001/025460 into a that include the steps of (l) electroporating target DNA ce of a plant that contains a first pore of the plant a recombinagenic oligonucleobase the sequence of at least 6 base pairs of homologous region that has a sequence identical to which has a DNA sequence and a second homologous region a first fragment of the target of the of at least 6 base pairs of a second fragment sequence identical to the sequence which contains at least 1 base target DNA ce, and an intervening region intervening region connects the first heterologous to the target DNA sequence, which (2) ing the microspore to homologous region and the second homologous region; the embryo a plant having a mutation located produce an embryo; and (3) producing from of the target DNA sequence, e. g., by culturing n the first and second nts and regenerating the plant from the . microspore to produce a somatic embryo is an MDON various embodiments of the invention, the recombinagenic oligonucleobase RNA segment of at least 6 pe and each of the homologous regions contains an nucleotides in length; the first and or nucleotides; the intervening region is at least 3 2‘~substituted ribonucleotides. second RNA segment contains at least 8 contiguous is the targeted modification of One of the major goals of biological research for delivery of genes into mammalian the genome. As noted above, although methods modification and/or homologous recombination cells are well developed, the frequency of As a result, the modification of is limited (Hanson et al., Mol Cell Biol 15:45—51 1995).

Numerous methods have been contemplated or genes is a time consuming s. between donor and genomic attempted to enhance modification and/or recombination exhibit low rates of modification and/or DNA. However, the present techniques often and/or recombination rate, thereby recombination, or inconsistency in the modification hampering research and gene y technology.

SUMMARY OF THE INVENTION and compositions for The present ion provides novel methods modifications to specific locations in genomic improving the efficiency of the targeting of direct As described hereinafter, nucleic acids which or other nucleotide sequences. combined with various approaches to enhance the specific changes to the genome may be in the cells being targeted availability of components of the natural repair systems present for modification. 2014/029566 In a first aspect, the ion relates to methods for introducing a gene repair oligonucieehase (GRON—mediated mutation into a target ibonucleic acid (DNA) cell under ce in a plant cell. The methods comprise, inter alia, culturing the plant conditions that increase one or more cellular DNA repair processes prior to, and/or coincident with, delivery of a GRON into the plant cell; and/or ry of a GRON into the plant cell greater than 55 bases in length, the GRON optionally comprising two or more mutation sites for introduction into the target DNA.

In certain embodiments, the conditions that increase one or more cellular DNA repair processes comprise one or more of: introduction of one or more sites into the GRON or into the plant cell DNA that are targets for base excision repair, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for non- homologous end g, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for microhomology-mediated end g, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for homologous recombination, and introduction of one or more sites into the GRON or into the plant cell DNA that are targets for pushing repair.

As described hereinafter, GRONs for use in the present invention can comprises one or more of the following alterations from conventional RNA and DNA nucleotides: one or more abasic nucleotides; one or more 8’oxo dA and/or 8’0X0 dG nucleotides; a reverse base at the 3’ end thereof; one or more 2’ O—methyl nucleotides; one or more 2’O—methyl RNA nucleotides at the 5’ end thereof, and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; an alating dye; a 5’ terminus cap; a backbone modification selected from the group consisting of a phosphothioate modification, a methyl phosphonate modification, a locked nucleic acid (LNA) PCT/USZOl4/029566 modification, a O thoxyethyl) (MOE) modification, a di PS modification, and a peptide nucleic acid (PNA) cation; one or more intrastrand crosslinks; one or more fluorescent dyes conjugated thereto, prefereably at the 5’ or 3’ end of the GRON; and one or more bases which increase hybridization energy. This list is not meant to be limiting.

As described hereinafter, in certain embodiments GRON quality and conversion efficiency may be improved by synthesizing all or a portion of the GRON using nucleotide multimers, such as dimers, trimers, tetramers, etc improving its purity.

In certain embodiments, the target deoxyribonucleic acid (DNA) sequence is within the plant cell . The plant cell may be non—transgenic or enic, and the target DNA sequence may be a transgene or an endogenous gene of the plant cell.

In n embodiments, the conditions that se one or more cellular DNA repair processes comprise introducing one or more compounds which induce single or double DNA strand breaks into the plant cell prior to or coincident with delivering the GRON into the plant cell. Exemplary nds are described hereinafter.

The methods and compositions described herein are applicable to plants lly. By way of example only, a plant s may be selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfafa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field field pea, faba bean, lentils, turnip, rutabaga, brussel s, lupin, cauliflower, kale, beans, poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, and lily. These may also apply in whole or in part to all other biological systems ing but not limited to bacteria, fungi and mammalian cells and even their organelles (e.g., mitochondria and chloroplasts), In certain embodiments, the methods further comprise rating a plant having a seeds mutation introduced by the GRON from the plant cell, and may comprise collecting from the plant.

In related s, the present invention relates to plant cells sing a genomic modification introduced by a GRON according to the methods described herein, a plant methods sing a genomic modification introduced by a GRON according to the described herein, or a seed comprising a genomic modification introduced by a GRON according to the methods bed herein. detailed Other embodiments of the invention will be apparent from the following description, exemplary embodiments, and .

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 s BFP to GFP conversion mediated by phosphothioate (PS) labeled labeled GRONs.

GRONs (having 3 PS moieties at each end of the GRON) and 5'Cy3/ 3'idC "Okazaki Fig. 2 depicts GRONS comprising RNA/DNA, ed to herein as Fragment GRONS." [0022a] Fig 3 depicts the native complex and the chimera reproduced from Cong et al., (2013) Science, Vol. 339 (6120), pp 819-823. [0022b] Fig 4 depicts a schematic of the expression vector for chimeric chNA.

DETAILED DESCRIPTION OF THE ION mediated by ped over the past few years, targeted genetic modification alteration oligonucleotides has been shown to be a valuable technique for use in the specific These of short stretches ofDNA to create deletions, short insertions, and point mutations. methods involve DNA pairing/annealing, followed by a DNA repair/recombination event. double—stranded DNA in a First, the nucleic acid anneals with its complementary strand in the factors. This annealing creates a centrally located process mediated by cellular protein mismatched base pair (in the case of a point mutation), resulting in a structural perturbation second step in the that most likely ates the endogenous protein machinery to te the their repair s: site—specific modification of the chromosomal sequence and even s the organelles (e.g., mitochondria and chloroplasts). This newly introduced mismatch revision of the DNA repair machinery to perform a second repair event, leading to the final novel approaches which target site. The present methods improve these methods by providing increase the PCT/U82014/029566 and reproducibility availability of DNA repair components, thus increasing the efficiency of gene repair—mediated modifications to targeted nucleic acids. tions defined To facilitate understanding of the invention, a number of terms are below. ic acid sequence,” “nucleotide sequence” and “polynucleotide and fragments or sequence” as used herein refer to an oligonucleotide or polynucleotide, which may be portions thereof, and to DNA or RNA of genomic or synthetic origin strand. single— or double—stranded, and represent the sense or antisense refer to a As used herein, the terms “oligonucleotides” and mers” about 201 nucleic acid sequence of at least about 10 nucleotides and as many as about 20—25 nucleotides, preferably about 15 to 30 nucleotides, and more preferably nucleotides, which can be used as a probe or er. used The terms odifying molecule” and “DNA—modifying reagent” as to a herein refer to a le which is capable of recognizing and specifically binding of modifying a target c acid sequence in the genome of a cell, and which is capable nucleotide sequence within the , wherein the recognition and specific binding the DNA—modifying molecule to the nucleic acid sequence is protein—independent. with a DNA—modifying term “protein—independent” as used herein in connection the presence and/or le means that the DNA—modifying molecule does not require ty of a protein and/or enzyme for the recognition of, and/or specific binding to, but not limited to nucleic acid sequence. DNA—modifying molecules are exemplified, and oligonucleotides triplex forming oligonucleotides, peptide nucleic acids, polyamides, molecules of the which are intended to promote gene conversion. The DNA—modifying which are used for ion are guished from the prior art‘s nucleic acid sequences 1987] in homologous recombination [Wong & Capecchi, Molec. Cell. Biol. 72294-2295, recombination that the prior art's c acid sequences which are used for homologous as used herein in connection with a are protein—dependent. The term “protein-dependent” of a protein and/or molecule means that the molecule requires the presence and/or activity and/or ic binding of the molecule to, a nucleic acid enzyme for the recognition of, whether a DNA—modifying molecule requires the sequence. Methods for determining and/or enzyme for the recognition of, and/or specific presence and/or activity of a protein PCT/U82014/029566 binding to, a nucleic acid sequence are within the skill in the art [see, e. g., Dennis et al.

Nucl. Acids Res. 27:4734—4742, 1999]. For example, the DNA—modifying molecule may be incubated in vitro with the nucleic acid ce in the absence of any proteins and/or molecule and enzymes. The detection of specific binding between the DNA—modifying the nucleic acid sequence demonstrates that the DNA—modifying molecule is protein- independent. On the other hand, the absence of ic binding between the DNA- ing molecule and the nucleic acid sequence demonstrates that the DNA—modifying molecule is protein-dependent and/or requires additional factors.

“Triplex forming ucleotide” (TF0) is defined as a sequence of DNA or RNA that is capable of binding in the major grove of a duplex DNA or RNA helix to form a triple helix. Although the TFO is not limited to any particular , a preferred length of the TFO is 200 nucleotides or less, more preferably 100 nucleotides or less, yet more preferably from 5 to 50 nucleotides, even more ably from 10 to 25 nucleotides, and most preferably from 15 to 25 nucleotides. gh a degree of formation of sequence specificity between the TFO and the duplex DNA is necessary for the triple helix, no ular degree of specificity is required, as long as the triple helix is capable of forming. Likewise, no specific degree of y or affinity between the TPO and the duplex helix is ed as long as the triple helix is capable of forming. While not intending to limit the length of the nucleotide sequence to which the TFO specifically binds in one embodiment, the nucleotide sequence to which the TFO specifically binds is from 1 to 100, more preferably from 5 to 50, yet more preferably from 10 to 25, and most preferably from 15 to 25, nucleotides. Additionally, e helix” is defined as a double- helical nucleic acid with an oligonucleotide bound to a target sequence within the double— helical nucleic acid. The “double—helical” nucleic acid can be any double—stranded nucleic acid including double—stranded DNA, double—stranded RNA and mixed duplexes of DNA and RNA. The double—stranded nucleic acid is not limited to any particular . However, in preferred embodiments it has a length of r than 500 bp, more ably greater than 1 kb and most preferably greater than about 5 kb. In many applications the double—helical nucleic acid is cellular, genomic c acid. The triplex forming oligonucleotide may bind to the target sequence in a parallel or anti-parallel manner.

“Peptide Nucleic Acids,” “polyamides” or “PNA” are nucleic acids wherein the phosphate backbone is replaced with an N—aminoethylglycine—based polyamide 2014/029566 natural for complementary nucleic acids than their structure. PNAs have a higher affinity rules. PNAs can form highly counter parts ing the Watson~Crick airing stoichiometry: (PNA)2.DNA. stable triple helix structures with DNA of the following limited to any particular gh the e nucleic acids and polyamides are not acids and ides is 200 nucleotides length, a preferred length of the peptide nucleic from 5 to 50 or less, and most preferably or less, more preferably 100 nucleotides of the nucleotide sequence to nucleotides long. While not intending to limit the length binds, in one embodiment, the which the peptide nucleic acid and polyamide specifically acid and polyamide specifically bind is nucleotide sequence to which the peptide nucleic from 5 to 25, and most from 1 to 100, more preferably from 5 to 50, yet more preferably preferably from 5 to 20, nucleotides. “cells” refers to a population The term “cel ” refers to a single cell. The term one cell type. Likewise, of cells. The population may be a pure population comprising cell type. In the present invention, there is the population may comprise more than one that a cell population may comprise. no limit on the number of cell types when referring to a sample of The term “synchronize” or “synchronized,” cell population” refers to a plurality of cells, or “synchronized cells” or “synchronized of cells to be in the same phase of cells which have been treated to cause the population the sample be synchronized. A the cell cycle. It is not necessary that all of the cells in with the majority of the cells in the small percentage of cells may not be synchronized is between lO—lOO%. A more sample. A preferred range of cells that are synchronized preferred range is between 30—lOO%. Also, it is not necessary that the cells be a pure be contained in the . population of a single cell type. More than one cell type may be synchronized or may be in a different phase In this regard, only one of cell types may in the sample. of the cell cycle as compared to another cell type reference to a single cell means The term “synchronized cell” when made in it is at a cell cycle phase which is different that the cell has been manipulated such that the manipulation. Alternatively, a from the cell cycle phase of the cell prior to to alter (i.e., increase or “synchronized cel ” refers to a cell that has been manipulated the cell was prior to the decrease) the on of the cell cycle phase at which cell in the absence of the manipulation when compared to a l cell (e. g., a manipulation).

PCT/USZOl4/029566 The term “cell cycle” refers to the physiological and morphological progression of changes that cells undergo when dividing (i.e. proliferating). The cell 37 ‘5 cycle is generally recognized to be composed of phases termed “interphase, prophase,” “metaphase,” “anaphase,” and “telophase”. Additionally, parts of the cell cycle may be termed “M (mitosis),” “S (synthesis),” “G0,” “G1 (gap 1)” and “G2 (gap2)”.

Furthermore, the cell cycle includes periods of progression that are intermediate to the above named phases.

The term “cell cycle tion” refers to the cessation of cell cycle progression in a cell or population of cells. Cell cycle inhibition is usually induced by re of the cells to an agent (chemical, proteinaceous or otherwise) that interferes with aspects of cell physiology to prevent continuation of the cell cycle.

“Proliferation” or “cell growth” refers to the ability of a parent cell to divide into two daughter cells repeatably y resulting in a total increase of cells in the population. The cell population may be in an organism or in a culture apparatus.

The term “capable of modifying DNA” or “DNA modifying means” refers to procedures, as well as endogenous or exogenous agents or reagents that have the ability to induce, or can aid in the induction of, changes to the nucleotide sequence of a targeted segment of DNA. Such changes may be made by the deletion, addition or tution of one or more bases on the targeted DNA segment. It is not necessary that the DNA ce changes confer functional changes to any gene encoded by the targeted sequence. Furthermore, it is not necessary that changes to the DNA be made to any particular portion or percentage of the cells.

The term “nucleotide sequence of st” refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any , by one of ordinary skill in the art. Such tide sequences include, but are not limited to, coding ces of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug ance genes, growth factors, etc.), and non—coding regulatory sequences that do not encode an mRNA or protein product (e.g., promoter sequence, enhancer sequence, polyadenylation sequence, ation sequence, regulatory RNAs such as miRNA, etc.).

“Amino acid sequence,3) peptide sequence,’9 ‘6peptide ce” and “peptide” are used interchangeably herein to refer to a sequence of amino acids. 2014/029566 double-helical c acid “Target sequence,” as used herein, refers to a in length but less than 201 comprising a sequence preferably greater than 8 nucleotides between 8 nucleotides in length. In some ments, the target ce is preferably is defined by the nucleotide sequence on one to 30 bases. The target sequence, in general, of the strands on the double-helical nucleic acid. when As used herein, a “purine-rich sequence” or “polypurine sequence” of the strands of a double—helical made in reference to a nucleotide sequence on one of nucleotides n r nucleic acid sequence is defined as a contiguous sequence n a purine base. However, it is than 50% of the nucleotides of the target sequence than 60% purine preferred that the purine—rich target sequence contain greater next most preferably nucleotides, more preferably greater than 75% purine tides, and most preferably 100% purine nucleotides. greater than 90% purine tides As used herein, a “pyrimidine-rich sequence” or “polypyrimidine sequence” of the strands of a double—helical when made in reference to a nucleotide sequence on one of nucleotides wherein r nucleic acid sequence is defined as a contiguous sequence contain a pyrimidine base. r, it that 50% of the nucleotides of the target sequence contain greater than 60% pyrimidine is preferred that the pyrimidine—rich target sequence nucleotides. In some nucleotides and more preferably greater than 75% dine 3 than 90% pyrimidine nucleotides embodiments, the sequence contains preferably greater nucleotides. and, in other embodiments, is most preferably 100% pyrimidine nucleotide sequence A “variant” of a first nucleotide sequence is defined as a or more deletions, which differs from the first nucleotide sequence (e. g., by having one or using DNA insertions, or substitutions that may be detected using hybridization assays cing). Included within this definition is the detection of alterations or For e, modifications to the genomic sequence of the first nucleotide sequence. in the pattern of restriction hybridization assays may be used to detect (1) alterations of izing to the first nucleotide sequence when enzyme fragments e of a selected portion of the comprised in a genome (i.e., RFLP analysis), (2) the inability of genomic DNA which contains the first nucleotide sequence to hybridize to a sample (3) improper first nucleotide sequence (e.g., using allele—specific oligonucleotide probes), than the normal as hybridization to a locus other or unexpected hybridization, such fluorescent in situ chromosomal locus for the first nucleotide sequence (e.g., using PCT/USZOl4/029566 hybridization (FISH) to metaphase chromosomes spreads, etc.). One example of a variant is a mutated wild type sequence.

The terms “nucleic acid” and “unmodified nucleic acid” as used herein refer to any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, ne, and thymine). The term “modified c acid” refers to a nucleic acid whose structure is altered relative to the structure of the fied nucleic acid. Illustrative of such modifications would be replacement covalent modifications of the bases, such as alkylation of amino and ring nitrogens as well as saturation of double bonds.

As used herein, the terms “mutation” and “modification” and grammatical equivalents thereof when used in reference to a nucleic acid sequence are used interchangeably to refer to a deletion, insertion, substitution, strand break, and/or introduction of an adduct. A “deletion” is defined as a change in a c acid sequence in which one or more nucleotides is . An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides. A “substitution” s from the ement of one or more nucleotides by a molecule which is a different le from the replaced one or more nucleotides. For example, a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine. Pyrimidine to pyrimidine (e. g. C to T or T to C nucleotide substitutions) or purine to purine (eg. G to A or A to G nucleotide substitutions) are termed transitions, whereas pyrimidine to purine or purine to pyrimidine (e.g. G to T or G to C or A to T or A to C) are termed transversions. Alternatively, a nucleic acid may be replaced by a modified c acid as ified by replacement of a thymine by thymine glycol. Mutations may result in a mismatch. The term “mismatch” refers to a non—covalent interaction between two c acids, each nucleic acid ng on a different polynucleic acid sequence, which does not follow the base-pairing rules.

For example, for the partially complementary sequences 5'—AGT~3’ and 5’~AAT—3’, a GA mismatch (a transition) is present. The terms “introduction of an adduct” or “adduct formation” refer to the covalent or non-covalent linkage of a molecule to one or more nucleotides in a DNA ce such that the linkage results in a reduction (preferably from 10% to 100%, more preferably from 50% to 100%, and most preferably from 75% to 100%) in the level of DNA replication and/or transcription.

The term “strand break” when made in reference to a double stranded nucleic acid sequence includes a single-strand break and/or a -strand break. A single— of the two strands of the double strand break (a nick) refers to an interruption in one refers ed nucleic acid sequence. This is in st to a double-strand break which double stranded nucleic acid sequence. Strand to an interruption in both strands of the nucleic acid sequence either directly breaks may be introduced into a double stranded certain chemicals) or indirectly (e.g., by (e. g., by ionizing radiation or ent with tic incision at a nucleic acid base). refer to a cell which contains at The terms “mutant cell” and “modified cell” least one modification in the cell‘s genomic sequence. nucleotide sequence refers to The term “portion” when used in nce to a in size from 5 fragments of that nucleotide sequence. The fragments may range minus one nucleic acid residue. nucleotide residues to the entire nucleotide sequence ends” because DNA molecules are said to have “5’ ends” and “3’ such that the 5’ mononucleotides are reacted to make oligonucleotides in a manner attached to the 3’ oxygen of its neighbor ate of one mononucleotide pentose ring is in one direction via a phosphodiester linkage. ore, an end of an oligonucleotide is linked to the 3’ oxygen of a referred to as the “5’ end” if its 5’ phosphate is not is referred to as the “3’ end” mononucleotide pentose ring. An end of an oligonucleotide of another mononucleotide pentose ring. if its 3’ oxygen is not linked to a 5’ ate if internal to a larger oligonucleotide, also As used herein, a nucleic acid sequence, even discrete and 3’ ends. In either a linear or circular DNA molecule, may be said to have 5' ’ of the “downstream” or 3’ elements. elements are referred to as being “upstream” or in a 5’ to 3’ direction along the DNA This terminology reflects that ription proceeds direct transcription of a linked gene . The promoter and enhancer elements which of the coding region. However, enhancer elements are generally located 5’ or am 3’ of the promoter element and the coding . can exert their effect even when located located 3’ or downstream of the Transcription termination and polyadenylation signals are coding region. herein refers to a DNA The term “recombinant DNA molecule” as used means of molecular molecule which is comprised of segments of DNA joined together by biological techniques. as used herein The term “recombinant protein” or “recombinant polypeptide” recombinant DNA molecule. refers to a n molecule which is expressed using a PCT/USZOl4/029566 As used herein, the terms “vector” and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to The terms “in operable combination,9’ 44' in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the sis of a desired protein molecule is produced. The terms also refer to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

The term fection” as used herein refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate—DNA co—precipitation, DEAE-dextran—mediated transfection, polybrene—mediated transfection, electroporation, microinjection, liposome fusion, lipofectin, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.

As used herein, the terms ementary” or ementarity” are used in nce to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the ce “5’—CAGT—3’,” is complementary to the sequence “5’~ACTG-3’.” Complementarity can be a ” or “tota ”. “Partial” complementarity is where one or more nucleic acid bases is not matched ing to the base pairing rules. “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands. This may be of ular importance in amplification reactions, as well as detection methods which depend upon g between nucleic acids. For the sake of ience, the terms “polynucleotides” and “oligonucleotides” include molecules which include nucleosides.

The terms “homology” and “homologous” as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be l homology or complete homology (i.e., identity). When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term “substantially homologous” refers to any nucleic acid sequence (e. g., PCT/U82014/029566 probe) which can hybridize to either or both strands of the -stranded nucleic acid sequence under conditions of low stringency as described above. A nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid from sequence is one that at least partially ts a completely complementary sequence hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely mentary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low ency A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely gous sequence to a target sequence under conditions of low stringency. This is not to say that ions of low stringency are such that non~specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e. g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non—complementary target.

Low stringency conditions comprise conditions equivalent to binding or ization at 68° C. in a solution consisting of SXSSPE (43.8 g/l NaCl, 6.9 g/l NaHZPO4°HZO and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardt's reagent (50x Denhardt's contains per 500 m1: 5 g Ficoll (Type 400, cia), 5 g BSA (Fraction V; Sigma» and 100 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising 2.0xSSPE, 0.1% SDS at room temperature when a probe of about 100 to about 1000 nucleotides in length is employed.

In addition, conditions which promote hybridization under ions of high stringency (e. g., sing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) are well known in the art. High stringency conditions, when used in reference to nucleic acid hybridization, comprise conditions equivalent to g or ization at 68°C. in a solution consisting of SXSSPE, 1% SDS, ardt‘s reagent and 100 rig/ml denatured salmon sperm DNA followed by washing in a on comprising 0.1xSSPE and 0.1% SDS at 68°C when a probe of about 100 to about 1000 nucleotides in length is employed.

It is well known in the art that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature PCT/USZOI4/029566 (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in on or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization on may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.

The term “equivalent” when made in reference to a hybridization condition as it relates to a hybridization condition of st means that the hybridization ion and the hybridization condition of interest result in hybridization of nucleic acid For example, if a sequences which have the same range of t (%) homology. hybridization condition of st results in hybridization of a first nucleic acid sequence with other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization ion of interest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence.

As used herein, the term “hybridization” is used in nce to the pairing of complementary c acids using any process by which a strand of c acid joins with a complementary strand h base pairing to form a ization complex.

Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, ency of the conditions involved, the Tm of the formed hybrid, and the SC ratio within the nucleic acids.

As used herein the term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization x may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e. g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, ing FISH (fluorescent in situ hybridization».

PCT/U82014/029566 reference to the “melting As used herein, the term “Tm” is used in which a population of temperature.” The melting temperature is the temperature at iated into single strands. The double-stranded nucleic acid molecules becomes half is well known in the art. As indicated by equation for ating the Tm of nucleic acids be calculated by the equation: rd references, a simple estimate of the Tm value may in s solution at l M NaCl (see e.g., Tm=8l .5+0.4l(% G+C), when a nucleic acid is in Nucleic Acid Anderson and Young, Quantitative Filter Hybridization, which Hybridization,l985). Other references include more sophisticated computations into account for the calculation of Tm. take structural as well as sequence characteristics used in reference to the conditions of As used herein the term “stringency” is of other compounds such as organic temperature, ionic th, and the presence ted. “Stringency” typically solvents, under which nucleic acid hybridizations are As will be to about 20°C. to 25°C. below Tm. occurs in a range from about Tm°C. can be used to identify or understood by those of skill in the art, a stringent hybridization or detect similar or related detect identical polynucleotide sequences or to identify polynucleotide sequences. g and grammatical i [0064] The terms “specific binding,” “binding icity,” E of a first tide sequence equivalents thereof when made in reference to the binding the first to the preferential interaction n to a second nucleotide sequence, refer to the interaction nucleotide ce with the second nucleotide sequence as compared third nucleotide sequence. Specific between the second nucleotide sequence with a absolute icity of g; in other binding is a relative term that does not require that the second nucleotide sequence words, the term “specific binding” does not require in the absence of an interaction between interact with the first nucleotide sequence Rather, it is sufficient that second nucleotide sequence and the third nucleotide sequence. and the second nucleotide the level of interaction between the first nucleotide sequence nucleotide sequence the level of interaction between the second sequence is r than with with the third nucleotide sequence. “Specific binding” of a first nucleotide sequence first nucleotide also means that the interaction between the a second nucleotide sequence of a nucleotide sequence is dependent upon the presence sequence and the second in other words the second particular structure on or within the first nucleotide sequence; structure on or within the nucleotide sequence is recognizing and binding to a specific acids or to nucleotide sequences in first nucleotide ce rather than to nucleic WO 44951 general. For example, if a second nucleotide sequence is specific for structure “A” that is on or within a first nucleotide sequence, the presence of a third nucleic acid sequence containing ure A will reduce the amount of the second nucleotide sequence which is bound to the first nucleotide sequence.

As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids which may be amplified by any ication method. It is plated that “amplifiable nucleic acid” will usually comprise “sample template.” The terms “heterologous nucleic acid sequence” or “heterologous DNA” are used interchangeably to refer to a nucleotide sequence which is ligated to a c acid different location sequence to which it is not ligated in nature, or to which it is ligated at a in . Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e. g., proteins which confer drug resistance), etc.

“Amplification” is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art enbach C W and G S Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). As used , the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat.

Nos. 4,683,195, and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the ied segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter.

By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target ce become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR ied.” With PCR, it is possible to amplify a single copy of a specific target sequence in c DNA to a level detectable by l different methodologies (e.g., PCT/U82014/029566 of biotinylated primers followed by hybridization with a labeled probe; incorporation of 32P—labeled deoxynucleotide —enzyme conjugate ion; incorporation segment). In addition to triphosphates, such as dCTP or dATP, into the amplified be amplified with the appropriate set of genomic DNA, any oligonucleotide sequence can the PCR process itself primer molecules. In particular, the amplified segments created by for subsequent PCR amplifications. are, themselves, efficient templates for commercial applications, is based One such preferred method, ularly real~time PCR technology, and es Allele—Specific on the widely used TaqMan® of the wildype allele.

PCR with a Blocking reagent (ASB-PCR) to suppress amplification somatic mutations in either DNA or ASB—PCR can be used for ion of germ line or formalin—fixed paraffin—embedded RNA ted from any type of tissue, including rules are developed enabling sensitive tumor specimens. A set of reagent design insertions, or deletions against a selective detection of single point substitutions, (Morlan J, Baker J, background of wild-type allele in thousand—fold or greater excess.

A Simple, Robust and Highly Sinicropi D Mutation Detection by ime PCR: Selective . PLoS ONE 4(2): e4584, 2009) chain reaction” and “RT—PCR” The terms “reverse transcription rase of an RNA sequence to generate a mixture refer to a method for reverse transcription concentration of a desired segment of the cDNA sequences, followed by increasing the or purification, Typically, transcribed cDNA sequences in the mixture without cloning an oligo—dT primer) prior to RNA is reverse transcribed using a single primer (e. g., DNA using two primers. ication of the d t of the ribed whether As used herein, the term “primer” refers to an oligonucleotide, synthetically, which is ing naturally as in a purified restriction digest or produced when placed under conditions in capable of acting as a point of initiation of synthesis which is complementary to a nucleic acid which synthesis of a primer extension product of an inducing agent such as strand is induced, (i.e., in the presence of nucleotides and and pH). The primer is preferably single DNA polymerase and at a suitable temperature but may alternatively be double stranded for maximum efficiency in amplification, its strands before being stranded. If double stranded, the primer is first treated to separate used to prepare extension products. Preferably, the primer is an the synthesis of oligodeoxyribonucleotide. The primer must be sufficiently long to prime The exact lengths of the primers extension products in the ce of the inducing agent.

PCT/U82014/029566 will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a ed restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double—stranded.

Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e. g., ELISA, as well as —based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the t invention be limited to any particular detection system or label.

As used herein, the terms “restriction endonucleases” and iction enzymes” refer to ial enzymes, each of which cut or nick double— or single—stranded DNA at or near a specific nucleotide sequence, for e, an endonuclease domain of a type 118 restriction endonuclease (e.g., Fold) can be used, as taught by Kim etal., 1996, Proc, Nat’l. Acad. Sci. USA, 6:1 156—60).

As used herein, the term “an oligonucleotide having a nucleotide sequence encoding a gene” means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, c DNA or RNA form. When present in a DNA form, the oligonucleotide may be —stranded (i.e., the sense strand) or double-stranded.

Additionally “an oligonucleotide having a tide sequence encoding a gene” may include suitable control elements such as enhancers, promoters, splice junctions, polyadenylation signals, etc. if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Further still, the coding region of the present invention may contain endogenous enhancers, splice ons, intervening sequences, polyadenylation signals, etc.

Transcriptional l signals in eukaryotes comprise “enhancer” elements.

Enhancers consist of short arrays of DNA sequences that interact specifically with cellular ns involved in transcription (Maniatis, T. et al., Science 236: 1237, 1987).

Enhancer elements have been ed from a variety of eukaryotic sources including W0 2014/144951 genes in plant, yeast, insect and mammalian cells and viruses. The selection of a particular enhancer depends on what cell type is to be used to express the protein of interest.

The presence of “splicing signals” on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the l of introns from the primary RNA transcript and consist of a splice donor and acceptor site ook, J. et a1., Molecular Cloning: A tory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp. 16.7—16.8, 1989). A commonly used splice donor and acceptor site is the splice junction from the 168 RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the ent ation and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.

The term “poly A site” or “poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript.

Efficient enylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be “heterologous” or “endogenous.” An endogenous poly A signal is one that is found lly at the 3’ end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3’ of another gene.

The term “promoter,” “promoter element” or “promoter sequence” as used herein, refers to a DNA sequence which when placed at the 5’ end of (i.e., precedes) an oligonucleotide sequence is capable of controlling the transcription of the oligonucleotide sequence into mRNA. A promoter is typically located 5’ (Le, upstream) of an oligonucleotide sequence whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and for initiation of transcription.

The term “promoter activity” when made in nce to a c acid of an sequence refers to the ability of the c acid sequence to initiate transcription oligonucleotide sequence into mRNA.

The term e specific” as it applies to a er refers to a er that is capable of ing selective expression of an oligonucleotide sequence to a specific PCT/U82014/029566 of the same oligonucleotide in a type of tissue in the relative absence of expression different type of tissue. Tissue specificity of a promoter may be evaluated by, for example, ly linking a reporter gene to the promoter sequence to generate a reporter of a plant or an animal such construct, ucing the reporter construct into the genome that the reporter constmct is integrated into every tissue of the resulting transgenic animal, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, in different tissues of the or the activity of a protein d by the reporter gene) enic plant or animal. Selectivity need not be te. The detection of a greater the level of level of expression of the reporter gene in one or more tissues relative to for the expression of the reporter gene in other tissues shows that the promoter is ic tissues in which greater levels of expression are detected.

The term “cell type specific” as applied to a promoter refers to a promoter in a which is capable of directing selective expression of an oligonucleotide sequence specific type of cell in the relative absence of expression of the same ucleotide within the same tissue. The term “cell type specific” sequence in a different type of cell selective when applied to a er also means a promoter capable of promoting expression of an oligonucleotide in a region within a single tissue. Again, selectivity need not be absolute. Cell type specificity of a promoter may be assessed using methods herein. well known in the art, e. g., immunohistochemical staining as described Briefly, reacted with a primary tissue sections are embedded in paraffin, and paraffin sections are antibody which is specific for the polypeptide t encoded by the oligonucleotide the promoter. As an alternative to in sequence whose expression is lled by sectioning, samples may be cryosectioned. For example, sections may be frozen prior to A labeled and during sectioning thus avoiding ial interference by residual paraffin. for the primary (e. g., peroxidase ated) secondary antibody which is specific detected (e.g., antibody is allowed to bind to the sectioned tissue and specific binding with avidin/biotin) by microscopy.

The terms “selective sion,” “selectively express” and grammatical in two or more equivalents thereof refer to a comparison of relative levels of expression in connection with s of st. For e, “selective expression” when used of interest in a tissues refers to a substantially greater level of expression of a gene the gene particular tissue, or to a substantially greater number of cells which express of, and the number within that tissue, as compared, respectively, to the level of expression PCT/U52014/029566 of cells sing, the same gene in another tissue (i.e., selectivity need not be absolute).

Selective expression does not require, although it may include, expression of a gene of interest in a particular tissue and a total absence of expression of the same gene in another tissue. Similarly, “selective expression” as used herein in nce to cell types refers to a substantially greater level of expression of, or a ntially greater number of cells which express, a gene of interest in a ular cell type, when compared, respectively, to the expression levels of the gene and to the number of cells expressing the gene in another cell type.

The term guous” when used in reference to two or more nucleotide sequences means the nucleotide sequences are d in tandem either in the absence of ening sequences, or in the presence of intervening sequences which do not comprise one or more control elements.

As used herein, the terms ic acid molecule encoding, 57 ‘Cnucleotide encoding,” “DNA sequence encoding” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is separated from at least one contaminant nucleic acid with which it is ordinarily ated in its natural source.

Isolated nucleic acid is nucleic acid present in a form or setting that is ent from that in which it is found in nature. In st, non—isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific n, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a polypeptide of interest includes, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide of interest where the nucleic acid is in a chromosomal or extrachromosomal location ent from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single—stranded or double—stranded form. Isolated nucleic acid can be readily identified (if d) by a variety of techniques (e.g., hybridization, dot blotting, etc.). When an ed nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single—stranded). Alternatively, it may contain both the sense and anti—sense s (i.e., the oligonucleotide may be double—stranded).

As used , the term “purified” or “to purify” refers to the removal of one or more ired) components from a sample. For example, where inant polypeptides are expressed in bacterial host cells, the polypeptides are purified by the removal of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample.

As used herein, the term “substantially purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free and more preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” is, therefore, a substantially purified polynucleotide.

As used herein the term g region” when used in reference to a structural gene refers to the nucleotide ces which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5’ side generally by the tide triplet “ATG” which encodes the initiator nine and on the 3’ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

By ”coding sequence" is meant a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment f. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

By "non—coding sequence" is meant a sequence of a nucleic acid or its complement, or a part thereof that is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non—coding sequences e both intron sequences in genomic DNA or immature primary RNA transcripts, and ssociated sequences such as ers, enhancers, silencers, etc.

PCTfUSZOl4/029566 As used , the term “structural gene” or tural nucleotide sequence” refers to a DNA ce coding for RNA or a protein which does not control the expression of other genes. In contrast, a “regulatory gene” or “regulatory sequence” is a structural gene which encodes products (e.g., transcription factors) which control the expression of other genes.

As used herein, the term “regulatory element” refers to a genetic element which controls some aspect of the expression of nucleic acid ces. For example, a promoter is a regulatory element which facilitates the tion of transcription of an operany linked coding region. Other regulatory elements include splicing s, polyadenylation signals, termination signals, etc.

As used herein, the term “peptide transcription factor binding site” or “transcription factor binding site” refers to a nucleotide sequence which binds protein transcription factors and, thereby, controls some aspect of the expression of nucleic acid sites are examples of sequences. For e, Sp—l and APl (activator protein 1) g peptide transcription factor binding sites.

As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene. A “gene” may also include non- translated sequences located adjacent to the coding region on both the 5’ and 3’ ends such that the gene ponds to the length of the full—length mRNA. The sequences which are located 5’ of the coding region and which are present on the mRNA are ed to ’ non-translated sequences. The sequences which are located 3’ or downstream of the coding region and which are present on the mRNA are referred to as 3’ non—translated forms of a gene. A sequences. The term “gene” encompasses both cDNA and genomic genomic form or clone of a gene contains the coding region upted with non-coding Introns sequences termed “introns” or “intervening s” or “intervening sequences.” are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or ed out” from the nuclear or primary transcript; introns therefore are absent in the translation to y messenger RNA (mRNA) transcript. The mRNA functions during the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include E which are present on the sequences located on both the 5’ and 3’ end of the sequences PCT/U82014/029566 RNA transcript. These sequences are referred to as “flanking” sequences or regions (these g sequences are located 5’ or 3’ to the non—translated ces present on the mRNA transcript). The 5’ flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3’ g region may contain sequences which direct the termination of transcription, post— transcriptional cleavage and polyadenylation.

A “non—human animal” refers to any animal which is not a human and includes vertebrates such as rodents, non—human primates, ovines, bovines, ruminants, lagomorphs, es, caprines, equines, canines, felines, aves, etc. Preferred non—human animals are selected from the order Rodentia. “Non—human animal” additionally refers to amphibians (e. g. Xenopus), es, insects (e.g. Drosophila) and other non—mammalian animal species.

As used herein, the term “transgenic” refers to an organism or cell that has DNA derived from another sm inserted into which becomes integrated into the genome either of somatic and/or germ line cells of the plant or animal. A “transgene” means a DNA sequence which is partly or ly heterologous (i.e., not present in nature) to the plant or animal in which it is found, or which is homologous to an endogenous ce (i.e., a sequence that is found in the animal in nature) and is inserted into the plant’ or 's genome at a location which differs from that of the naturally occurring sequence. Transgenic plants or animals which include one or more transgenes are within the scope of this invention. Additionally, a “transgenic” as used herein refers to an animal that has had one or more genes modified and/or “knocked out” (made non—functional or made to function at reduced level, i.e., a “knockout” mutation) by the invention‘s methods, by homologous recombination, TFO on or by similar processes. For example, in some embodiments, a transgenic organism or cell includes inserted DNA that includes a foreign promoter and/or coding region.

A “transformed cell” is a cell or cell line that has acquired the ability to grow in cell culture for le generations, the ability to grow in soft agar, and/or the ability to not have cell growth inhibited by cell—to—cell t. In this regard, transformation refers to the introduction of foreign genetic material into a cell or organism.

Transformation may be lished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. “Transformation” includes but is not limited to such methods as W0 2014/144951 PCT/USZOl4/029566 transfection, microinjection, electroporation, nucleofection and lipofection (liposome— mediated gene transfer). Transformation may be accomplished through use of any expression vector. For example, the use of baculovirus to introduce n nucleic acid into insect cells is contemplated. The term “transformation” also includes methods such as P—element mediated germline transformation of whole s. Additionally, transformation refers to cells that have been transformed lly, usually through genetic mutation.

As used herein “exogenous” means that the gene encoding the protein is not normally sed in the cell. onally, “exogenous” refers to a gene transfected into a cell to augment the normal (i.e. natural) level of expression of that gene.

A peptide ce and nucleotide sequence may be “endogenous” or “heterologous” (i.e., “foreign”). The term “endogenous” refers to a sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification relative to the naturally-occurring sequence. The term “heterologous” refers to a sequence which is not endogenous to the cell into which it is introduced. For example, heterologous DNA includes a nucleotide sequence which is ligated to, or is lated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide ce which is naturally found in the cell into which it is introduced and which ns some modification relative to the naturally—occurring sequence. Generally, although not necessarily, heterologous DNA encodes heterologous RNA and heterologous proteins that are not normally produced by the cell into which it is introduced. Examples of logous DNA include er genes, riptional and translational regulatory sequences, DNA sequences which encode able marker proteins (e.g., proteins which confer drug resistance), etc.

Constructs The nucleic acid molecules sed herein (e.g., site specific nucleases, or guide RNA for CRISPRs) can be used in the production of recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the t disclosure can be used in the preparation of nucleic acid constructs, for example, expression cassettes for expression in the plant of interest. This expression may be transient for instance when the construct is not integrated into the host genome or maintained under the control offered by the promoter and the position of the construct within the host’s genome if it becomes integrated.

Expression cassettes may include regulatory sequences operably linked to the site specific se or guide RNA sequences disclosed herein. The cassette may additionally contain at least one additional gene to be co-transformed into the organism atively, the additional ) can be provided on multiple sion cassettes.

The nucleic acid constructs may be provided with a plurality of restriction sites for insertion of the site ic nuclease coding ce to be under the transcriptional regulation of the regulatory regions. The nucleic acid constructs may additionally contain nucleic acid molecules encoding for selectable marker genes.

Any promoter can be used in the production of the nucleic acid constructs.

The promoter may be native or ous, or n or heterologous, to the plant host c acid ces disclosed herein. Additionally, the promoter may be the natural is “foreign” or sequence or alternatively a synthetic sequence. Where the promoter “heterologous” to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding ce.

The site directed nuclease sequences disclosed herein may be expressed using heterologous promoters.

] Any promoter can be used in the preparation of constructs to control the expression of the site directed nuclease sequences, such as promoters providing for constitutive, tissue—preferred, inducible, or other promoters for expression in plants.

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WC 99/43 838 and US. Patent No. 6,072,050; the core CaMV 358 promoter (Odell et al. Nature 313:810—8 12; 1985); rice actin (McElroy et al., Plant Cell 22163—171, 1990); ubiquitin (Christensen et al., Plant M01. Biol. 12:619-632, 1989 and Christensen et al., Plant Mol. Biol. 18:675—689, 1992); pEMU (Last et al., Theor. Appl. Genet. 81:581—588, 1991); MAS (Velten eta1., EMBO J. 3:2723-2730, 1984); ALS promoter (US. Patent No. 026), and the like. Other constitutive promoters include, for example, US. Patent Nos. 5,608,149; 5,608,144; ,604,121; 5,569,597; 5,466,785; 680; 5,268,463; 5,608,142; and 6,177,611.

WO 44951 Tissue-preferred promoters can be utilized to direct site directed nuclease expression within a particular plant tissue. Such tissue—preferred promoters include, but are not limited to, leaf—preferred promoters, root-preferred promoters, seed—preferred promoters, and stem~preferred promoters. Tissue-preferred promoters include Yamamoto eta1., Plant J. 255-265, 1997; Kawamata et al., Plant Cell Physiol. 38(7):792—803, 1997', Hansen eta1., M01. Gen Genet. 254(3):337—343, 1997; Russell eta1., Transgenic Res. 6(2):157-168, 1997; Rinehartet al., Plant l. 1 12(3):1331-1341, 1996; Van Camp et al., Plant l. 1 12(2):525—535, 1996; Canevascini et al., Plant Physiol. 112(2): 513-524, 1996; to et al., Plant Cell l. 35(5):773—778, 1994; Lam, Results Probl. Cell Differ. 20:181—196, 1994; Orozco et al. Plant M01 Biol. 23(6)11129— 1138, 1993; Matsuoka et al., Proc Nat’l. Acad. Sci. USA 90(20):9586~ 9590, 1993; and Guevara—Garcia et al., Plant J. 4(3):495—505, 1993.

The nucleic acid constructs may also include transcription termination regions.

Where transcription terminations regions are used, any termination region may be used in the preparation of the nucleic acid constructs. For example, the ation region may be derived from another source (i.e., foreign or heterologous to the promoter). Examples of termination regions that are available for use in the constructs of the present disclosure include those from the Ti—plasmid of A. tzmzefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau eta1., Mol. Gen. Genet. 262:141—144, 1991; Proudfoot, Cell 64:671-674, 1991; Sanfacon et al., Genes Dev. 149, 1991; Mogen et al., Plant Cell 2:1261~1272, 1990; Munroe et al., Gene 91:151—158, 1990;Ba11as eta1., Nucleic Acids Res. 17:7891~7903, 1989; and Joshieta1., Nucleic Acid Res. 15:9627—9639, 1987.

In ction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the nucleic acids may be optimized for increased expression in the ormed plant. That is, the c acids encoding the site directed nuclease proteins can be synthesized using plant—preferred codons for improved expression. See, for example, Campbell and Gowri, (Plant Physiol. 1, 1990) for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant—preferred genes. See, for example, US. Patent Nos. 5,380,831, and ,436,391, and Murray et al., Nucleic Acids Res. 17:477—498, 1989.

In addition, other ce modifications can be made to the nucleic acid sequences disclosed herein. For example, additional sequence modifications are known to enhance gene expression in a cellular host. These e elimination of sequences ng spurious polyadenylation signals, exon/intron splice site signals, transposon—like repeats, and other such well—characterized sequences that may be deleterious to gene expression. The SC content of the sequence may also be adjusted to levels average for a target cellular host, as calculated by reference to known genes sed in the host cell.

In on, the sequence can be modified to avoid predicted hairpin secondary mRNA StI'UClLUI’CS.

Other nucleic acid sequences may also be used in the preparation of the constructs of the present disclosure, for example to enhance the sion of the site directed nuclease coding sequence. Such nucleic acid sequences include the introns of the maize Adhl, intronl gene (Callis et al., Genes and Development 1:1183—1200, 1987), and leader sequences, (W—sequence) from the Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallic et al., Nucleic Acid Res. :8693—871 1, 1987; and Skuzeski et al., Plant Mol. Biol. 15:65—79, 1990). The first intron from the shrunken—1 locus of maize has been shown to increase expression of and 5,593,874 disclose the genes in chimeric gene constructs. US. Pat. Nos. 5,424,412 use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106929-9259, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize site ed nuclease gene expression, the plant expression vectors disclosed herein may also n DNA sequences containing matrix attachment regions (MARS). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the disclosure.

The expression constructs disclosed herein can also include nucleic acid ed se sequence to the sequences capable of directing the sion of the site chloroplast. Such c acid sequences include chloroplast targeting sequences that encodes a chloroplast transit peptide to direct the gene product of interest to plant cell chloroplasts. Such transit peptides are known in the art. With respect to chloroplast— ‘2 targetng sequences, “operably ” means that the c acid sequence encoding a i transit peptide (i.e., the chloroplast-targeting sequence) is linked to the site directed l nuclease nucleic acid molecules disclosed herein such that the two sequences are uous and in the same reading frame. See, for e, Von Heijne et al., Plant l Mol. Biol. Rep. 9:104—126, 1991; Clark et al., J. Biol. Chem. 264:17544—17550, 1989; Della—Cioppa et a1., Plant Physiol. 84:965—968, 1987; Romer et a1., Biochem. Biophys.

Res. Commun. 14—1421, 1993; and Shah et al., e 233:478—481, 1986.

Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose—1,5‘bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et a1., Plant Mol. Biol. —780, 1996; Schnell et a1., J. Biol. Chem. 266(5):3335~3342, 1991); 5— (enolpyruvyl)shikimate—3—phosphate synthase (EPSPS) (Archer et al., J. Bioenerg. Biomemb. 22(6):789—810, 1990); tryptophan synthase (Zhao et al., J. Biol. Chem. 270(1 1):6081— 6087, 1995); plastocyanin (Lawrence et a1., J. Biol.

Chem. 272(33):20357~20363, 1997); chorismate synthase (Schmidt et a1., J. Biol. Chem. 268(3 6):27447—27457, 1993); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et a1., J. Biol. Chem. 263: 14996-14999, 1988). See also Von Heijne et a1., Plant Mol. Biol. Rep. 9: 104—126, 1991; Clark et a1., J. Biol. Chem. 264:17544—17550, 1989; Della-Cioppa et a1., Plant Physiol. 84:965~968, 1987; Romer et a1., Biochem.

Biophys. Res. Commun. 196:1414-1421, 1993; and Shah et al., e 233 2478—481, 1986.

In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the c acid constructs may be ed to direct the expression of the mutant site directed se coding sequence from the plant cell chloroplast. Methods for transformation of chloroplasts are known in the art. See, for example, Svab et a1., Proc. Nat’l. Acad. Sci. USA 87:8526—8530, 1990; Svab and Maliga, Proc. Nat’l. Acad. Sci. USA 90:913—917, 1993; Svab and Maliga, EMBO J. 12:601—606, 1993. The method relies on particle gun delivery of DNA containing a able marker and targeting of the DNA to the plastid genome through homologous recombination.

Additionally, d transformation can be accomplished by transactivation of a silent plastid—borne ene by tissue—preferred expression of a nuclear-encoded and plastid— directed RNA polymerase. Such a system has been reported in McBride et al. Proc.

Nat’l. Acad. Sci. USA 91:7301—7305, 1994.

The nucleic acids of interest to be targeted to the plast may be optimized for expression in the chloroplast to account for ences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for e, US. Patent No. ,380,831, herein incorporated by reference.

PCT/USZOI4/029566 ] The nucleic acid ucts can be used to transform plant cells and regenerate transgenic plants comprising the site directed nuclease coding sequences. Numerous plant transformation vectors and methods for transforming plants are available. See, for example, US. Patent No. 6,753,458, An, G. et al., Plant Physiol., 81:301—305, 1986; Fry, J. et al., Plant Cell Rep. 6:321—325, 1987; Block, M., Theor. Appl Genet. 76:767-774, 1988; Hinchee eta1., r. Genet. Symp.203212.203—212, 1990; Cousins et al., Aust. J.

Plant Physiol. 18:481—494, 1991; Chee, P. P. and Slightom, J. L., Gene.118:255—260, 1992; Christou et al., Trends. Biotechnol. 10:239—246, 1992; D'Halluin et al., Bio/Techno]. —3 14, 1992; Dhir et al., Plant Physiol. 88, 1992; Casas eta1., Proc. Nat’l. Acad Sci. USA 90:11212—11216, 1993; Christou, P., In Vitro Cell. Dev.

Biol—Plant 29P:1 19124, 1993; Davies, et al., Plant Cell Rep. 12:180-183, 1993; Dong, J.

A. and Mc Hughen, A., Plant Sci. 91:139—148, 1993; in, C. I. and Trieu, T. N., Plant. Physiol. 102:167, 1993; Golovkin et al., Plant Sci. 90:41-52, 1993', Guo Chin Sci.

Bull. 38:2072~2078; Asano, eta1., Plant Cell Rep. 13, 1994; Ayeres N. M. and Park, W.

D., Crit. Rev. Plant. Sci. 13:219-239, 1994; Barcelo et al., Plant. J. 5:583—592, 1994; Becker, et al., Plant. J. 5:299-307, 1994; Borkowska et al., Acta. Physiol Plant. 16:225- 230, 1994; Christou, P., Agro. Food. Ind. Hi Tech. 5:17—27, 1994; Eapen eta1., Plant Cell Rep. 13:582—586, 1994; Hartman et al., Bio—Technology 923, 1994; Ritala et al., Plant. M01. Biol. 24:317—325, 1994; and Wan, Y. C. and Lemaux, P. (3., Plant Physiol. 10423748, 1994. The constructs may also be ormed into plant cells using homologous ination.

The term “wi1d~type” when made in reference to a peptide ce and tide sequence refers to a peptide sequence and nucleotide ce (locus/gene/allele), respectively, which has the characteristics of that peptide sequence and nucleotide sequence when isolated from a naturally occurring source. A wild—type peptide sequence and nucleotide sequence is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wi1d~type” form of the peptide sequence and nucleotide sequence, respectively. "Wiltl~type" may also refer to the sequence at a specific nucleotide position or positions, or the sequence at a particular codon on or positions, or the sequence at a particular amino acid position or positions.

“Consensus ce” is defined as a sequence of amino acids or nucleotides that contain identical amino acids or nucleotides or functionally equivalent amino acids or PCT/USZOI4/029566 nucleotides for at least 25% of the sequence. The identical or functionally equivalent amino acids or nucleotides need not be contiguous The term “Brassica” as used herein refers to plants of the Brassica genus.

Exemplary Brassica species include, but are not limited to, B. carinata, B. elongate, B. fruticulosa, B. , B. napus, B. narinosa, B. nigra, B. ea, B. perviridis, B. rapa (syn B. tris), B. rupestris, B. septiceps, and B. tournefortii.

A nucleobase is a base, which in certain preferred embodiments is a purine, pyrimidine, or a derivative or analog thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2'—deoxyriboside.

Nucleosides can be linked by one of several linkage moieties, which may or may not contain phosphorus. Nucleosides that arc linked by unsubstituted phosphodiester linkages are termed nucleotides. The term "nucleobase" as used herein includes peptide nucleobases, the subunits of peptide c acids, and morpholine nucleobases as well as sides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; preferably at least a portion of which can hybridize by Watson—Crick base pairing to a DNA having the complementary sequence. An oligonucleobase chain may have a single 5' and 3' terminus, which are the ultimate nucleobases of the r. A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that may be complementary and ized by Watson—Crick base g. Ribo-type bases include pentosefuranosy] containing bases wherein the 2‘ carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type bases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.

In certain embodiments, an oligonucleobase strand may include both oligonucleobase chains and segments or regions of ucleobase chains. An oligonucleobase strand may have a 3‘ end and a 5‘ end, and when an oligonucleobase strand is coextensive with a chain, the 3‘ and 5‘ ends of the strand are also 3' and 5‘ termini of the chain. 2014/029566 The term ”gene repair oligonucleobase" as used herein denotes oligonucleobases, including mixed duplex oligonucleotides, non—nucleotide containing molecules, single stranded oligodeoxynucleotides and other gene repair molecules.

As used herein the term “codon” refers to a sequence of three adjacent tides r RNA or DNA) constituting the genetic code that detemiines the insertion of a specific amino acid in a polypeptide chain during n synthesis or the signal to stop protein synthesis. The term "codon" is also used to refer to the ponding (and complementary) sequences of three nucleotides in the messenger RNA into which the original DNA is transcribed.

As used herein, the term "homology" refers to sequence similarity among proteins and DNA. The term "homology” or ”homologous" refers to a degree of identity.

There may be partial homology or complete homology. A partially homologous sequence is one that has less than l00% sequence identity when ed to another sequence.

"Heterozygous" refers to having different alleles at one or more genetic loci in homologous chromosome segments. As used herein "heterozygous" may also refer to a sample, a cell, a cell population or an sm in which different alleles at one or more genetic loci may be detected. Heterozygous samples may also be determined via s known in the art such as, for example, nucleic acid sequencing. For example, if a sequencing opherogram shows two peaks at a single locus and both peaks are roughly the same size, the sample may be characterized as heterozygous. Or, if one peak is smaller than another, but is at least about 25% the size of the larger peak, the sample may be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the r peak is at least about % of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

As used herein, "homozygous" refers to having identical s at one or more genetic loci in homologous chromosome segments. ”Homozygous" may also refer to a sample, a cell, a cell tion or an organism in which the same alleles at one or more genetic loci may be detected. Homozygous samples may be determined via methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a particular locus, the sample may be termed "homozygous" with respect to that locus.

PCT/U82014/029566 The term ”hemizygous" refers to a gene or gene t being present only once in the genotype of a cell or an organism because the second allele is deleted. As used herein "hemizygous" may also refer to a sample, a cell, a cell population or an organism in which an allele at one or more c loci may be detected only once in the genotype.

The term ”zygosity status" as used herein refers to a sample, a cell population, or an organism as appearing heterozygous, gous, or hemizygous as determined by testing methods known in the art and described herein. The term ”zygosity status of a nucleic acid" means determining whether the source of nucleic acid appears heterozygous, homozygous, or hemizygous. The ity status" may refer to differences in a single nucleotide in a sequence. In some methods, the zygosity status of a sample with respect to a single mutation may be categorized as homozygous wild—type, heterozygous (i.e., one wild-type allele and one mutant allele), homozygous mutant, or hemizygous (i.e., a single copy of either the wild—type or mutant ).

As used herein, the term "RTDS" refers to The Rapid Trait Development SystemTM (RTDS) developed by Cibus. RTDS is a site—specific gene modification system that is ive at making precise changes in a gene sequence without the incorporation of foreign genes or control sequences.

The term "about" as used herein means in quantitative terms plus or minus %. For example, "about 3%" would encompass 27—33% and "about 10%" would encompass 9—] l %.

Repair Oligonucleotides This invention generally relates to novel methods to improve the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences. onally, this invention relates to target DNA that has been modified, mutated or marked by the approaches disclosed herein. The ion also relates to cells, , and organisms which have been modified by the invention's methods. The present invention builds on the development of compositions and methods related in part to the successful sion system, the Rapid Trait pment System (RTDSTM, Cibus US LLC).

RTDS is based on altering a targeted gene by utilizing the cell‘s own gene repair system to ically modify the gene sequence in situ and not insert foreign DNA and gene expression control sequences. This procedure effects a precise change in the PCT/U82014/029566 genetic sequence while the rest of the genome is left unaltered. In contrast to conventional transgenic GMOs, there is no integration of foreign genetic material, nor is any foreign genetic material left in the plant. The changes in the genetic ce introduced by RTDS are not randomly inserted. Since affected genes remain in their native location, no , uncontrolled or adverse pattern of expression occurs.

The RTDS that effects this change is a ally sized oligonucleotide which may be composed of both DNA and modified RNA bases as well as other chemical moieties, and is designed to hybridize at the targeted gene location to create a mismatched base—pair(s). This mismatched base~pair acts as a signal to attract the cell‘s own natural gene repair system to that site and correct (replace, insert or ) the designated nucleotide(s) within the gene. Once the correction process is complete the RTDS molecule is degraded and the now—modified or repaired gene is expressed under that gene‘s normal nous control mechanisms.

The methods and compositions disclosed herein can be ced or made with " chemistries as gene repair oligonucleobases" (GRON) having the conformations and described in detail below. The ” gene repair oligonucleobases" as plated herein have also been described in published scientific and patent literature using other names including ”recombinagenic oligonucleobases;" "RNA/DNA chimeric oligonucleotides;” "chimeric oligonucleotides;” "mixed duplex oligonucleotides" (MDONs); "RNA DNA I! II I! ll oligonucleotides (RDOs);" " gene targeting oligonucleotides; genoplasts; single stranded modified oligonucleotides;" "Single stranded oligodeoxynucleotide mutational vectors“ (SSOMVs); x mutational vectors;” and "heteroduplex mutational vectors." The gene repair oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers stic delivery), microfibers, hylene glycol (PEG)—mediated uptake, oporation, and microinj ection.

In one embodiment, the gene repair oligonucleobase is a mixed duplex oligonucleotides (MDON) in which the RNA—type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by ing the 2‘~hydroxyl with a fluoro, chloro or bromo onality or by placing a substituent on the 2'~O. Suitable substituents include the substituents taught by the Kmiec 11. Alternative substituents include the tuents taught by US. Pat. No. 5,334,71 l (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin W0 2014/144951 PCT/U82014/029566 Applications), which are hereby incorporated by reference. As used herein, a 2'—fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a T- OH substituted with a substituent described in the Martin Applications or Sproat is termed a "T- Substituted Ribonucleotide." As used herein the term ype nucleotide" means of a mixed a T~ hydroxyl or 2 ‘—Substituted Nucleotide that is linked to other nucleotides duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non— natural linkages taught by Kmiec I or Kmiec II. As used herein the term "deoxyribo—type nucleotide" means a nucleotide having a T—H, which can be linked to other tides of of the a gene repair oligonucleobase by an tituted phosphodiester linkage or any non—natural es taught by Kmiec I or Kmiec II.

In a particular embodiment of the present invention, the gene repair oligonucleobase is a mixed duplex oligonucleotide (MDON) that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, odiester tives and non-phosphorus-based linkages as taught by Kmiec II. In yet another embodiment, each pe nucleotide in the mixed duplex oligonucleotide is a 2 ‘-Substituted Nucleotide. Particular preferred embodiments of 2‘-Substituted Ribonucleotides are 2'-fluoro, T~ methoxy, pyloxy, 2'—allyloxy, 2'—hydroxylethyloxy, hoxyethyloxy, T— fluoropropyloxy and 2'- trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2'— Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy, and 2'— allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted odiester bonds, Although mixed duplex ucleotides (MDONs) having only a single type of 2'— substituted pe tide are more conveniently synthesized, the methods of the invention can be practiced with mixed duplex oligonucleotides having two or more be affected by types of RNA—type nucleotides. The on of an RNA segment may not an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses terms such as "interrupted RNA segment." An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA segment can contain alternating RNase- resistant and unsubstituted 2’—OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson—Crick WO 44951 PCT/U82014/029566 base paired. In one embodiment the strands of the mixed duplex oligonucleotide are ntly bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3' and a single 5‘ end. The 3' and 5' ends can be ted by the addition of a "hairpin cap" whereby the 3' and 5' al nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3’ and 5' ends, so that the Watson- Crick pairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologous with two fragments of the target gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA— type nucleotides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a "heterologous region." The heterologous region can contain one, two or three mismatched nucleotides. The ched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides.

Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer tides from the mixed duplex oligonucleotide. The length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region.

The distance between the fragments of the target gene that are complementary to the two homologous regions is cal to the length of the heterologous region where a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous s are thereby ted in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, Le, a region that is identical in sequence to a fragment of the target gene, which ts er preferably contain at least 13 RNA-type nucleotides PCTfUS2014/029566 preferably from 16 to 25 pe nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., "connected by" an intervening DNA segment. In one ment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a "mutator segment." In another embodiment of the present invention, the gene repair oligonucleobase (GRON) is a single stranded oligodeoxynucleotide mutational vector (SSOMV), which is disclosed in International Patent Application PCT/USOO/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 which is incorporated by reference in its entirety. The sequence of the SSOMV is based on the same principles as the mutational vectors described in US. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; ,888,983; 5,795,972; 5,780,296; 339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/5 8702; and WO 99/40789. The ce of the SSOMV contains two s that are homologous with the target ce ted by a region that contains the desired genetic alteration termed the mutator . The r region can have a sequence that is the same length as the ce that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such an SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one, two, or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of the target gene.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3‘ terminal and/or 5' terminal internucleotide linkage or alternatively the two 3' terminal and/or 5' terminal internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide e is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3' end nucleotide or 5 ‘ end nucleotide and a ng tuent. In a specific embodiment the length of the SSOMV is between 21 and 55 W0 2014/144951 PCT/USZOl4/029566 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding or the non— coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotide and the targeted nucleotide be a dine. To the extent that is consistent with achieving the desired functional result, it is preferred that both the r nucleotide and the targeted nucleotide in the mentary strand be pyrimidines. Particularly preferred are SSOMVs that encode transversion mutations, Le, a C or T mutator nucleotide is ched, respectively, with a C or T tide in the mentary strand. ing efficiency The present invention bes a number of ches to increase the effectiveness of conversion of a target gene using repair oligonucleotides, and which may be used alone or in combination with one another. These include: 1. Introducing cations to the repair oligonucleotides which attract DNA repair machinery to the targeted (mismatch) site.

A. Introduction of one or more abasic sites in the oligonucleotide (e.g, within bases, and more preferably with 5 bases of the desired mismatch site) generates a lesion which is an intermediate in base excision repair (BER), and which attracts BER machinery to the vicinity of the site targeted for conversion by the repair, oligonucleotide. dSpacer (abasic furan) modified oligonucleotides may be prepared as described in, for example, Takeshita et al., J. Biol. Chem, 262:10171—79, 1987.

B. Inclusion of nds which induce single or double strand breaks, either into the oligonucleotide or together with the oligonucleotide, generates a lesion which is repaired by non—homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination. By way of example,the bleomycin family of antibiotics, zinc fingers, Fokl (or any type IIS class of restriction enzyme) and other nucleases may be covalently coupled to the 3’ or 5’ end of repair clignnucleotides, order to introduce double strand breaks in the vicinity of the site targeted for WO 44951 PCT/USZOI4/029566 conversion by the repair ucleetide. The bleomycin family of antibiotics are DNA cleaving glycopeptides include bleomycin, zeocin, phleomycin, tallysomycin, pepleomycin and others.

C. Introduction of one or more 8’oxo dA or dG incorporated in the oligonucleotide (e. g., within 10 bases, and more preferably with 5 bases of the desired mismatch site) generates a lesion which is similar to s d by reactive oxygen species. These lesions induce the so-called “pushing repair” system. See, e.g., Kim et a1., Biochem. Mol. Biol. 372657—62, 2004.

, J. 2. Increase stability of the repair oiigonueleotides: Introduction of a e base (idC) at the 3’ end of the oligonucleotide to create a 3’ blocked end on the repair oiigonucleotide.

Introduction of one or more 2’O—Inethyl nucleotides or bases which increase ization energy (see, e. g., WO2007/O73l49) at the 5’ and/or 3’ of the repair oligonucieotide.

Introduction of a plurality of 2’0—methyl RNA nucleotides at the 5’ end of the repair ueleotide, leading into DNA bases which provide the desired mismatch site, thereby creating an Okazaki Fragment-like nucleic acid structure.

Conjugated (5’ or 3’) intercalating dyes such as acridine, psoralen, ethidium bromide and Syber stains.

Introduction of a 5’ us cap such as a T/A clamp, a cholesterol moiety, SIMA (HEX), riboC and amidite. ne modifications such as phosphothioate, 2’—O methyl, methyl phosphonates, locked nucleic acid (LNA), MOE (methoxyethyl), di PS and peptide nucleic acid (PNA).

Crosslinking of the repair oiigonucleotide, e.g, with intrastrand emsslinking reagents agents: such as cisplatin and mitomycin C.

Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5, Cy3B, Cy5 and DY647.

PCT/U82014/029566 3. Increase hybridization energy of the repair oligonucleotide through incorporation of bases which increase hybridization energy (see, e.g., WOZDO7/073l49). 4. Increase the quality of repair oligonueleotide synthesis by using nucleotide multimers (dimers, trimers, tetramers, etc.) as building blocks for synthesis. This results in fewer coupling steps and easier separation of the full length products from building blocks.

. Use of long repair oligonueleotides (ire, greater than 55 nucleotides in length, preferably between 75 and 300 nucleotides in length, more ably at least 100 nucleotides in length, still more preferably at least 150 tides in length, and most preferably at least 200 nucleotides in length), ably with two or more mutations targeted in the repair oli gonucieotide.

Examples of the foregoing approaches are provided in the following table ] Table 1. GRON chemistries to be tested.

Olige type Metiifisatiens ’ mods i/A clamp. TIA clamp Backbone cations Phosphothioate PS l'ntercalating dyes 5‘ Acn‘iiine ‘3‘ idC Acritiine, idC 01:33am fragments l— BNAJRNA CyB ements D37547 Facilitators Z'OMe oligos designed 5’ E‘O‘Me and 3' of the ting oligo Ahasic Ahasie site placed. in Abasic 2 various locations 3’ and 3’ to the converting base. 44 mer Assist Assist rsh {1373, hit: on one. none on (fiveriap: the other: 2 oiigos: l with {IyBIidCL l umnorlified repair oligo Assist Assist approach only make the utm‘iodified No overlap: oiigo 2 oligoe: l with CyBI’idC, i uimiodified repair oligo 2014/029566 {Riga iyge Mmiificaéiuns Abasic THE“ site. piaced in various "i“eirahydmfman {aispacer'} locations 5' and 3‘ :0 the. cum/mfing base. 414mm Backbcmfi modificatims 9 2'0Me "i‘fin‘mrs '.i"rimc:r amidites, C313. idC 1-H) shing repair fi’ 8‘0x0 silk, 5’ (:33? idii I 1 Pushing repair 8‘0x0 dA, 5 3'3, MC Double S‘u‘and break Bleomycin Cn‘msiinker {5.819.123 Lin.

CTOSSHI’IELEI‘ cin C Facilitators super bases 5’ and 3' of ’2 amino GA and, 2-— {bin '1‘ converting oligo Super oliggs 2mm: (3: d, 53‘ C373, MC Supm‘ 0} igos 2-thi0 T, 5' (3373, MC Super oligos a A, 5‘ (33.8, idC Super (xiigos 741621121 (3,5' CyS, idC Super eliggs pmpanyl (1C; 5’ Cy3, idC Intercalaimg dyas 5' Psmalanfl’ idC Paw-61.1w, idC {marmalatmg dyes E" Ethidium brmnida: Intercaiating dyes 5' Sybar stains ‘ mode 5‘ Che.- 3' MC (Emissmrol Baum: mutadm Long, afiiga (”i 00 bases) w/ Unin‘wwn ’2. mutation ' mods 5' SIM/X HEX/33$? SEMA HEX, MC Backbone modifications 9 Methyl phosphonates Backbmw modifications {NA ne. modifications 1 MQE {methc-xyethyl) W0 44951 Giige type Madiiieatiens (33/3 replacements Cy3 5 {33/3 replacements; CyS Backbone rimdificatimrs iii. PS ‘ mods. riboC for branch nm ne modifications PNA Cy3 repfiacenrents 'i.‘)’Y'6-—'17 ' mods 5' branch symmetric branch amidite/MC The foregoing modifications may also include known nucleotide modifications such as methylation, 5’ intercalating dyes, modifications to the 5’ and 3’ ends, ne modifiications, crosslinkers, cyclization and ‘caps' and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Modifications of nucleotides include the addition of acridine, amine, biotin, cascade blue, cholesterol, Cy3 @, Cy5 @, Cy5 .5@ Daboyl, genin, dinitrophenyl, Edans, 6-FAM, fluorescein, 3'— glyceryl, HEX, {RD-700, O, JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA—S", SE, BODIPY", Marina Blue@, Pacific Blue@, Oregon , Rhodamine Green@, Rhodamine Red@, Rhodol Green@ and Texas Red@. Polynucleotide backbone modifications include methylphosphonate, 2'-OMe- methylphosphonate RNA, phosphorothiorate, RNA, 2'-OMeRNA. Base modifications include o—dA, 2~aminopurine, 3'— (ddA), 3‘dA (cordycepin), a—dA, 8-Br~dA, 8~ oxo-dA, N6-Me-dA, abasic site (dSpacer), biotin dT, 2‘~OMe-5Me-C, 2‘-OMe- propynyl-C, 3'- dC), 3'- (ddC), 5~Br~dC, 5-l—duc, 5-Me—dC, S—F-dC, carboxy-dT, convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, a~ dG, 8-Br-dG, 8~ oxo-dG, O6—Me~dG, S6—DNP—dG, 4~methyl—indole, 5—nitroindole, 2'- OMe—inosine, 2’-dl, 06— phenyl—dl, 4—methyl—indole, 2'—deoxynebularine, 5—nitroindole, 2— aminopurine, dP (purine analogue), dK (pyrimidine analogue), 3~nitropyrrole, Z-thio-dT, 4—thio—dT, biotin-dT, carboxy—dT, 04—Me—dT, azol dT, 2'—OMe—propynyl—U, S—Br— dU, 2’—dU, 5~F—dU, 5-l—dU, 04~t1iazol dU. Said terms also encompass peptide nucleic acids , a DNA analogue in which the backbone is a pseudopeptide consisting of N— (2~aminoethyl)—glycine units rather than a sugar. PNAs mimic the behavior of DNA PCTfUSZOl4/029566 and bind complementary nucleic acid s. The neutral backbone of PNA s in stronger binding and greater icity than normally achieved. In addition, the unique chemical, physical and ical properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and sors.

Oligonucleobases may have nick(s), gap(s), modified nucleotides such as modified oligonucleotide backbones, abasic nucleotides, or other al moieties. In a further embodiment, at least one strand of the oligonucleobase includes at least one additional modified nucleotide, e.g., a ethyl modified tide such as a MOE (methoxyethyl), a nucleotide having a 5’-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2’—deoxy~2’—fluoro ed nucleotide, a 2’-deoxy— modified tide, a locked nucleotide, an abasic nucleotide (the nucleobase is g or has a yl group in place thereof (see, e. g., Glen Research, http://www.glenresearch.com/GlenReports/GRZl —14.html)), a 2’~amino—modified nucleotide, a 2’-alkyl—modified nucleotide, a morpholino nucleotide, a phosphoramidite, and a tural base comprising nucleotide. Various salts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphoro—dithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3’—alkylene phosphonates, 5’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl—phosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3’—5' linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein one or more ucleotide linkages is a 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3’ to 3’ linkage at the 3’—most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a yl group in place thereof). The most common use of a linkage inversion is to add a 3'—3‘ linkage to the end of an antisense oligonucleotide with a phosphorothioate backbone. The 3‘—3' linkage further stabilizes the antisense oligonucleotide to exonuclease degradation by creating an oligonucleotide with two 5'— OH ends and no 3'—OH end. Linkage inversions can be introduced into specific locations PCT/U82014/029566 during oligonucleotide synthesis through use of "reversed phosphoramidites". These reagents have the phosphoramidite groups on the 5‘—OH position and the dimethoxytrityl (DMT) protecting group on the 3'—OH position. ly, the DMT protecting group is on the 5'—OH and the phosphoramidite is on the 3‘-OH.

Examples of modified bases include, but are not limited to, 2—aminopurine, 2’- amino—butyryl pyrene—uridine, 2’—aminouridine, 2'~deoxyuridine, 2’—fluoro—cytidine, 2’- fluoro—uridine, 2,6—diaminopun'ne, 4-thio-uridine, o—u1idine, S—fluoro—cytidine, 5— fluorouridine, 5—indo—uridine, S—methyl-cytidine, inosine, N3—methyl—uridine, 7—deaza— guanine, 8—an1inohexyl—amino—adenine, 6—thio—guanine, 4-thio~thymine, Z—thio-thymine, —iodo—uridine, 5-iodo—cytidine, 8—bromo—guanine, 8—bromo—adenine, a—adenine, 7- diaza—guanine, 8-oxo—guanine, hydro-uridine, and 5-hydroxymethyl—uridine. These tic units are commercially available; (for example, purchased from Glen Research Company) and can be incorporated into DNA by chemical synthesis.

Examples of modification of the sugar moiety are 3’—deoxylation, 2’— fluorination, and osidation, however, it is not to be construed as being limited o. Incorporation of these into DNA is also possible by chemical synthesis.

Examples of the 5' end cation are 5’~amination, 5’-biotinylation, 5’- fluoresceinylation, 5’—tetrafluoro~fluoreceinyaltion, 5’—thionation, and 5’~dabsylation, however it is not to be construed as being d thereto.

Examples of the 3’ end modification are 3’—amination, 3’—biotinylation, 2,3- dideoxidation, 3’—thionation, 3’—dabsylation, 3’-carboxylation, and 3’~cholesterylation, however, it is not to be construed as being limited thereto.

] In one preferred ment, the oligonucleobase can contain a 5' blocking substituent that is attached to the 5‘ terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible. A variety of non—toxic substituents such as biotin, cholesterol or other steroids or a non~interca1ating cationic fluorescent dye can be used. ularly preferred reagents to make oligonucleobases are the reagents sold as Cy3TM and CySTM by Glen Research, Sterling Va. (now GE Healthcare), which are d phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3',3‘— tetramethyl sopropyl substituted indomonocarbocyanine and carbocyanine dyes, respectively. Cy3 is particularly preferred. When the indocarbocyanine is N oxyalkyl substituted it can be conveniently linked to the 5' terminal of the oligodeoxynucleotide as a phosphodiester with a 5‘ terminal phosphate. When the commercially available Cy3 phosphoramidite is used as directed, the resulting 5‘ modification consists of a blocking substituent and linker together which are a N— ypropyl, sphatidylpropyl 3,3,3',3'~tetramethyl indomonocarbocyanine.

Other dyes contemplated include RhodamineéG, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Att0565, AttoSSO 26, Cy3.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, mStrawberry and y.

In a preferred embodiment the indocarbocyanine dye is tetra tuted at the 3 and 3‘ ons of the indole rings. t limitations as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions is not critical.

The oligo designs herein described might also be used as more efficient donor templates in ation with other DNA editing or recombination technologies including, but not limited to, gene targeting using site-specific homologous recombination by zinc finger nucleases, Transcription Activator—Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats RS).

The present invention generally s to methods for the efficient cation of genomic cellular DNA and/or recombination of DNA into the genomic DNA of cells. Although not d to any particular use, the methods of the present invention are useful in, for example, introducing a modification into the genome of a cell for the purpose of determining the effect of the modification on the cell. For example, a modification may be introduced into the nucleotide sequence which encodes an enzyme to ine whether the modification alters the enzymatic activity of the enzyme, and/or determine the location of the enzyme's catalytic region. Alternatively, the cation determine may be introduced into the coding sequence of a nding protein to whether the DNA binding activity of the protein is d, and thus to delineate the particular DNA-binding region within the protein. Yet another alternative is to introduce a modification into a non—coding regulatory sequence (e.g., promoter, enhancer, regulatory RNA sequence (miRNA), etc.) in order to determine the effect of the modification on the level of expression of a second sequence which is operably linked to the non-coding regulatory sequence. This may be desirable to, for example, define the particular sequence which possesses regulatory activity.

PCTfUSZOl4/029566 One strategy for producing targeted gene disruption is through the generation of single strand or double strand DNA breaks caused by site—specific endonucleases.

Endonucleases are most often used for targeted gene disruption in sms that have traditionally been refractive to more conventional gene targeting methods, such as algae, plants, and large animal models, including humans. For example, there are currently human clinical trials ay involving zinc finger ses for the treatment and prevention of HIV infection. Additionally, endonuclease ering is currently being used in attempts to disrupt genes that produce undesirable phenotypes in crops.

] The homing endonucleases, also known as meganucleases, are sequence specific endonucleases that generate double strand breaks in genomic DNA with a high degree of specificity due to their large (e. g., >14 bp) cleavage sites. While the specificity of the homing endonucleases for their target sites allows for precise targeting of the induced DNA breaks, homing endonuclease cleavage sites are rare and the probability of finding a naturally occurring cleavage site in a targeted gene is low.

One class of cial endonucleases is the zinc finger cleases. Zinc finger endonucleases combine a non—specific cleavage domain, lly that of Fold endonuclease, with zinc finger protein domains that are engineered to bind to specific DNA sequences. The modular structure of the zinc finger endonucleases makes them a versatile platform for delivering site-specific double—strand breaks to the . One limitation of the zinc finger endonucleases is that low specificity for a target site or the presence of multiple target sites in a genome can result in off—target cleavage .

FokI endonuclease cleaves as a dimer, one strategy to prevent off-target cleavage events has been to design zinc finger domains that bind at adjacent 9 base pair sites.

TALENs are targetable nucleases are used to induce single- and double—strand breaks into specific DNA sites, which are then repaired by mechanisms that can be exploited to create sequence alterations at the cleavage site.

The fundamental building block that is used to engineer the DNA~binding region of TALENS is a highly conserved repeat domain derived from lly occurring TALES encoded by Xanthomonas spp. proteobacteria. DNA binding by a TALEN is ed by arrays of highly conserved 33—35 amino acid repeats that are flanked by additional TALE-derived domains at the amino—terminal and carboxy—terminal ends of the repeats.

W0 2014/144951 PCT/USZOI4/029566 These TALE s ically bind to a single base of DNA, the identity of which is determined by two hypervariable residues typically found at positions 12 and 13 of the repeat, with the number of repeats in an array corresponded to the length of the desired target nucleic acid, the identity of the repeat selected to match the target nucleic acid sequence. The target nucleic acid is preferably between 15 and 20 base pairs in order to maximize selectivity of the target site. Cleavage of the target nucleic acid typically occurs within 50 base pairs of TALEN binding. Computer programs for TALEN recognition site design have been described in the art. See, e.g., Cermak et al., c Acids Res. 2011 July; 39(12): 682.

Once designed to match the desired target sequence, TALENS can be expressed recombinantly and introduced into protoplasts as exogenous proteins, or expressed from a plasmid within the protoplast.

Another class of cial endonucleases is the engineered meganucleases.

Engineered homing cleases are generated by modifying the specificity of existing homing endonucleases. In one approach, variations are introduced in the amino acid sequence of naturally occurring homing endonucleases and then the resultant engineered homing endonucleases are ed to select functional proteins which cleave a targeted binding site. In another approach, chimeric homing endonucleases are engineered by combining the recognition sites of two different homing cleases to create a new recognition site composed of a half— site of each homing endonuclease.

Other difying molecules may be used in targeted gene recombination. For e, peptide nucleic acids may be used to induce modifications to the genome of the target cell or cells (see, e.g., US. Pat. No. 053, to Ecker, herein orated by reference). In brief, synthetic nucleotides comprising, at least, a partial peptide backbone are used to target a homologous genomic nucleotide sequence.

Upon binding to the double-helical DNA, or through a mutagen ligated to the peptide nucleic acid, modification of the target DNA sequence and/or recombination is d to take place. Targeting icity is determined by the degree of sequence gy between the targeting sequence and the genomic sequence.

Furthermore, the present invention is not limited to the particular methods which are used herein to execute modification of genomic sequences. Indeed, a number of methods are contemplated. For example, genes may be targeted using triple helix W0 2014/144951 PCT/U82014/029566 forming oligonucleotides (TFO). TFOs may be generated synthetically, for example, by PCR or by use of a gene synthesizer apparatus. Additionally, TFOs may be isolated from genomic DNA if suitable natural sequences are found. TFOs may be used in a number of limited to, ways, including, for example, by tethering to a mutagen such as, but not psoralen or chlorambucil (see, e.g., Havre et al., Proc Nat’l Acad Sci, U.S.A. 9027879- 7883, 1993; Havre et al., J Virol 67:7323-7331, 1993; Wang et al., Mol Cell Biol :1759-1768, 1995; Takasugi et al., Proc Nat’l Acad Sci, U.S.A. 88:5602—5606, 1991; Belousov et al., c Acids Res 25:3440—3444, 1997). Furthermore, for example, TFOs may be tethered to donor duplex DNA (see, e.g., Chan et al., J Biol Chem 272:11541—11548, 1999). TPCs can also act by binding with ient affinity to provoke error—prone repair (Wang et al., Science 271:802-805, 1996).

The invention's methods are not limited to the nature or type of DNA— modifying t which is used. For example, such DNA—modifying reagents release radicals which result in DNA strand breakage. Alternatively, the reagents alkylate DNA to form adducts which would block replication and transcription. In another alternative, the ts generate crosslinks or les that t cellular enzymes leading to strand breaks. Examples of difying reagents which have been linked to oligonucleotides to form TFOs include, but are not d to, indolocarbazoles, lene diimide (NDI), transplatin, bleomycin, analogues of cyclopropapyrroloindole, and phenanthodihydrodioxins. In particular, indolocarbazoles are topoisomerase I inhibitors. Inhibition of these enzymes results in strand breaks and DNA protein adduct formation [Arimondo eta1., Bioorganic and Medicinal Chem. 8, 777, 2000]. NDI is a photooxidant that can oxidize guanines which could cause mutations at sites of guanine residues [Nunez, et al., Biochemistry, 39, 6190, 2000]. Transplatin has been shown to react with DNA in a triplex target when the TFO is linked to the reagent. This reaction causes the formation of DNA adducts which would be mutagenic [Columbier, et al., Nucleic Acids Research, 24: 4519, 1996]. Bleomycin is a DNA breaker, widely used as a radiation mimetic. It has been linked to oligonucleotides and shown to be active as a breaker in that format [Sergeyev, Nucleic Acids ch 23, 4400, 1995; Kane, et al., Biochemistry, 34, 16715, 1995]. Analogues of ropapyrroloindole have been linked to TFOs and shown to alkylate DNA in a triplex target sequence. The alkylated DNA would then n al adducts which would be mutagenic [Lukhtanov, et al., Nucleic Acids Research, 25, 5077, 1997]. Phenanthodihydrodioxins are masked quinones that release radical species upon photoactivation. They have been linked to T1903 and have been shown to introduce breaks into duplex DNA on ctivation [Bendinskas et al., Bioconjugate Chem. 9, 555, 1998].

Other methods of inducing modifications and/or recombination are contemplated by the present invention. For example, another embodiment involves the ion of homologous recombination between an exogenous DNA fragment and the targeted gene (see e.g., Capecchi et al., Science 244:1288—1292, 1989) or by using peptide nucleic acids (PNA) with affinity for the targeted site. Still other methods include sequence specific DNA recognition and ing by polyamides (see e.g., Dervan et al., Curr Opin Chem Biol 3:688—693, 1999; Biochemistry 38:2143-2151, 1999) and the use ses with site specific activity (e,g., zinc finger proteins, TALENs, Meganucleases and/or s).

The t invention is not limited to any ular frequency of modification and/or ination. The invention‘s methods result in a frequency of modification in the target tide sequence of from 0.2% to 3%. Nonetheless, any frequency (i.e., between 0% and 100%) of modification and/0r recombination is contemplated to be within the scope of the present invention. The frequency of cation and/or ination is dependent on the method used to induce the modification and/or recombination, the cell type used, the specific gene targeted and the DNA mutating t used, if any. Additionally, the method used to detect the modification and/or recombination, due to limitations in the detection method, may not detect all occurrences of modification and/or recombination. Furthermore, some modification and/or recombination events may be silent, giving no detectable indication that the modification and/or recombination has taken place. The inability to detect silent modification and/or recombination events gives an artificially low estimate of modification and/or recombination. e of these reasons, and others, the invention is not limited to any particular modification and/or recombination frequency. In one embodiment, the frequency of modification and/or recombination is between 0.01% and 100%. In r embodiment, the frequency of modification and/or recombination is between 0.01% and 50%. In yet another embodiment, the frequency of modification and/or recombination is between 0.1% and 10%. In still yet another embodiment, the frequency of modification and/or recombination is between 0.1% and 5%.

The term “frequency of mutation” as used herein in reference to a population of cells which are treated with a DNA—modifying molecule that is capable of ucing a mutation into a target site in the cells' genome, refers to the number of cells in the d population which contain the on at the target site as ed to the total number of cells which are treated with the DNA—modifying molecule. For example, with respect to a population of cells which is treated with the DNA-modifying molecule TFO tethered to psoralen which is designed to introduce a mutation at a target site in the cells‘ genome, a frequency of mutation of 5% means that of a total of 100 cells which are treated with TFO-psoralen, 5 cells contain a mutation at the target site.

Although the present invention is not limited to any degree of precision in the modification and/or recombination of DNA in the cell, it is contemplated that some embodiments of the present invention require higher s of precision, depending on the desired result. For example, the specific sequence changes required for gene repair (e.g., particular base changes) require a higher degree of precision as compared to producing a gene knockout wherein only the disruption of the gene is necessary. With the methods of the present invention, achievement of higher levels of precision in modification and/or homologous recombination techniques is greater than with prior art methods. ] ry of Gene Repair ucleobases into Plant Cells Any ly known method used to transform a plant cell can be used for ring the gene repair Oligonucleobases. Illustrative methods are listed below. The present invention contemplates many methods to transfect the cells with the DNA— modifying reagent or reagents. Indeed, the present invention is not limited to any particular method. Methods for the introduction of DNA modifying reagents into a cell or cells are well known in the art and e, but are not limited to, microinjection, electroporation, passive adsorption, m phosphate—DNA co—precipitation, DEAE— dextran-mediated transfection, polybrene—mediated transfection, liposome fusion, lipofectin, nucleofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.

The use of metallic microcarriers (microspheres) for introducing large fragments of DNA into plant cells having ose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). US. Pat.

Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for ting them.

Specific conditions for using microcarriers in the methods of the present invention are described in International Publication WO 99/07865. In an rative technique, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaClz and 0.1 M spermidine are added in that order; the mixture gently agitated, for 10 minutes, e.g., by vortexing, for 10 s and then left at room temperature whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Good s can be obtained with a concentration in the adhering solution of 8—10 ug/uL microcarriers, 14—17 ug/mL mixed duplex ucleotide, 4 M CaClz and 18—22 mM spermidine. Optimal results were observed under the conditions of 8 ug/uL microcarriers, 16.5 ug/mL mixed duplex oligonucleotide, 1.3 M CaClz and 21 111M spermidine.

Gene repair ucleobases can also be introduced into plant cells for the practice of the present invention using microfibers to penetrate the cell wall and cell membrane. US. Pat. No. 5,302,523 to Coffee et a1 describes the use of silicon carbide fibers to facilitate transformation of suspension maize es of Black n Sweet.

Any mechanical technique that can be used to introduce DNA for ormation of a plant cell using microfibers can be used to deliver gene repair ucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repair oligonucleobase is as follows: Sterile microfibers (2 pg) are suspended in 150 LIL of plant culture medium containing about 10 ug of a mixed duplex oligonucleotide. A suspension culture is allowed to settle and equal s of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 h as is appropriate for the particular trait.

In an ative embodiment, the gene repair oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part.

The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf, according to techniques well known to those skilled in the art. See, e. g., Gallois et a1, 1996, in Methods in Molecular Biology 55289-107, Humana Press, Totowa, N.J.; Kipp et a1., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, NJ.

The protoplasts need not be ed in growth media prior to electroporation. Illustrative conditions for electroporation are 3. times.10. sup.5 protoplasts in a total volume of 0.3 mL with a concentration of gene repair oligonucleobase of between 0.6—4 ug/mL.

In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the membrane—modifying agent hylene glycol, according to techniques well known to those skilled in the art. In another alternative embodiment, the into gene repair oligonucleobases can be delivered by injecting it with a microcapillary plant cells or into protoplasts In an alternative embodiment, nucleic acids are embedded in microbeads composed of calcium alginate and taken up by plant protoplasts in the presence of the ne-modifying agent polyethylene glycol (see, e. g., Sone et a1., 2002, Liu et a1., 2004).

In an ative embodiment, c acids forzen in water and introduced into plant cells by bombardment in the form of articles (see, e.g., Gilmore, 1991, US. Patent 5,219,746; ar et al.).

In an alternative embodiment, nucleic acids attached to nanoparticles are introduced into intact plant cells by incubation of the cells in a suspension containing the nanoparticlethe (see, e.g., Pasupathy et a1., 2008) or by delivering them into intact cells h particle bomardment or into protoplasts by co—incubation (see, e.g., Tomey et a1., 2007).

] In an alternative embodiment, nucleic acids complexed with penetrating peptides and delivered into cells by co—incubation (see, e.g., Chugh et a1., 2008, WO 2008148223 Al; Eudes and Chugh.

In an ative embodiment, nucleic acids are introduced into intact cells through electroporation (see, e.g., He et a1., 1998, US 2003/0115641 A1, Dobres et al.).

In an alternative ment, nucleic acids are delivered into cells of dry embryos by soaking them in a solution with nucleic acids (by soaking dry embryos in (see, e.g., Tepfer et a1., 1989, Senaratna et a1., 1991 ).

Selection of Plants ZOl4/029566 In various embodiments, plants as sed herein can be of any species of dicotyledonous, monocotyledonous or gyrnnospermous plant, including any woody plant species that grows as a tree or shrub, any herbaceous s, or any species that produces edible fruits, seeds or vegetables, or any species that es colorful or aromatic flowers. For example, the plant maybe selected from a species of plant from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfafa, barley, sorghum, tomato, mango, peach, apple, pear, erry, banana, melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, faba bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turf and forage grasses, flax, d rape, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plants insofar as they are not already specifically mentioned.

Plants and plant cells can be tested for resistance or tolerance to an herbicide using commonly known methods in the art, e.g., by g the plant or plant cell in the presence of an herbicide and measuring the rate of growth as compared to the growth rate in the absence of the herbicide.

As used herein, ntially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild—type AHAS protein.

As used herein, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more development events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those ing in a corresponding plant, plant organ, plant tissue or plant cell expressing the ype protein.

In certain embodiments plant organs provided herein include, but are not limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, dons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or ns, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, ground tissues, ZOl4/029566 vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues. Plant cells include, but are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and protoplasts.

Plants are substantially "tolerant" to a relevant herbicide when they are ted to it and provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non-tolerant like plant. Such dose/response curves have "dose" plotted on the X—axis and "percentage kill", "herbicidal effect", etc., plotted on the . Tolerant plants will require more herbicide than non- tolerant like plants in order to produce a given herbicidal effect Plants that are substantially "resistant" to the herbicide t few, if any, necrotic, lytic, chlorotic or other lesions, when subjected to herbicide at concentrations and rates which are typically employed by the agrochemical ity to kill weeds in the field. Plants which are resistant to an herbicide are also tolerant of the herbicide.

Generation of plants Tissue culture of various tissues of plant species and regeneration of plants rom is known. For example, the propagation of a canola cultivar by tissue culture is described in any of the following but not limited to any of the following: Chuong et al., "A Simple Culture Method for ca hypocotyls Protoplasts,” Plant Cell s 424- 6, 1985; Barsby, T. L., et al., ”A Rapid and ent Alternative Procedure for the Regeneration of Plants from Hypocotyl Protoplasts of Brassica napus,” Plant Cell s (Spring, 1996); Kartha, K., et al., "In vitro Plant Formation from Stem Explants of Rape," Physiol. Plant, 31 :217~220, 1974; Narasimhulu, S., et al., "Species Specific Shoot Regeneration Response of Cotyledonary ts of cas," Plant Cell Reports (Spring 1988); Swanson, 13., "Microspore Culture in Brassica," Methods in Molecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

] Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of soybeans and ration of plants therefrom is well known and widely published. For example, reference may be had to Komatsuda, T. et al., "Genotype X Sucrose Interactions for Somatic Embryogenesis in Soybeans," Crop Sci. 31:333-337, 1991; Stephens, P. A., et al., "Agronomic Evaluation of Tissue—Culture~Derived Soybean Plants," Theor. Appl. Genet. 82:633-635, 1991; PCT/U82014/029566 Komatsuda, T. et al., "Maturation and Germination of Somatic Embryos as Affected by Sucrose and Plant Growth Regulators in Soybeans Glycine gracilis z and Glycine max (L) Merr." Plant Cell, Tissue and Organ Culture, 28:103-113, 1992; Dhir, S. et al., "Regeneration of Fertile Plants from Protoplasts of Soybean ne max L. Menu); Genotypic Differences in Culture Response," Plant Cell Reports 11:285—289, 1992; Pandey, P. et al., ”Plant Regeneration from Leaf and Hypocotyl Explants of Glycine wightii (W. and A.) VERDC. var. longicauda," Japan J. Breed. 42:1~5, 1992; and Shetty, K., et al., ”Stimulation of In Vitro Shoot genesis in Glycine max (Merrill) by Allantoin and Amides," Plant Science 81:245—251, 1992. The disclosures of US Pat.

No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and US. Pat. No. 200 issued Apr. 16, 1991 to Ranch et al., are hereby incorporated herein in their entirety by reference.

EXAMPLES Example 1: GRON length Sommer et al., (Mol Biotechnol. 332115—22, 2006) bes a reporter system for the detection of in viva gene conversion which relies upon a single nucleotide change to convert between blue and green fluorescence in green fluorescent protein (GFP) variants. This reporter system was adapted for use in the following experiments using Arabidopsis thaliana as a model species in order to assess efficiency of GRON conversion following modification of the GRON .

In short, for this and the subsequent examples an Arabidopsis line with multiple copies of a blue fluorescent protein gene was created by methods known to those skilled in the art (see, e. g., Clough and Brent, 1998). Root-derived meristematic tissue es were established with this line, which was used for protoplast isolation and culture (see, e.g., Mathur et al., 1995). GRON delivery into protoplasts was achieved through polyethylene glycol (PEG) mediated GRON uptake into protoplasts. A method using a l format, similar to that described by similar to that bed by Fujiwara and Kato (2007) was used. In the ing the protocol is briefly described. The s given are those applied to individual wells of a 96—well dish.

PCT/U82014/029566 1. Mix 6.25 gal of GRON (80 uM) with 25 pl ofArabidopsis BFP transgenic root meristematic tissue—derived protoplasts at 5xlO6 cells/ml in each well of a 96 well plate. 2. 31.25 ul of a 40% PEG solution was added and the protoplasts were mixed. 3. Treated cells were incubated on ice for 30 min. 4. To each well 200 pl of W5 solution was added and the cells mixed. 5. The plates were allowed to incubate on ice for 30 min allowing the protoplasts to settle to the bottom of each well. ] 6. 200 pl of the medim above the settled protoplasts was d. 7. 85 ul of culture medium (MSAP, see Mathur etal., 1995) was added. 8. The plates were incubated at room ate in the dark for 48 hours. The final concentration of GRON after adding culture medium is 8 uM.

Forty eight hours after GRON delivery samples were analyzed by flow cytometry in order to detect protoplasts whose green and yellow fluorescence is different from that of control protoplasts (BFPO indicates non—targeting GRONS with no change compared to the EFF target; C is the coding strand design and NC is the non—coding strand design). A single C to T nucleotide difference (coding strand) or G to A tide targeted mutation (non—coding strand) in the center of the BFP4 molecules. The green fluorescence is caused by the introduction of a targeted mutation in the EFF gene, ing in the synthesis of GFP. The results are shown in Figure 1.

The following table shows the sequence of exemplary r and r C 5’—3PS/ 3’—3PS GRONs designed for the conversion of a blue fluorescent protein (BFP) gene to green fluorescence. (3P8 indicates 3 phosphothioate linkages at each of the 5’ and 3’ oligo ends).

WO 44951 2014/029566 [1102131 Table l: : GRON Name Q")WO'7' 2“g9..n‘33?.Q40 me: an5a:21na WWNC JOE—mar unuvflunaufln G 44' / Tome 101411131 {3‘4" - TGC TGC . 1'13 AAG CAC '11'3 ACC‘: cec- TGG 13m AAG GTG GT tAcc. AGGC(3’13 use CAG GC‘vC ACG GGC‘. AGC. 1191:1313 '$*'1‘*G ., .T":3>'4,C 1.1"'-:1mer ‘1 1 . . Ah 1TT1 1 CTA CGG LGT GCA (3:G CTTCAG CC‘G CTA (31:1, CGAC'CA CAT G'AA 131A 131A 1-313% \\\\\‘\\\\\\\\\\\\\\\\\\\\\.\\\.\.\\\\\\\\\\\.\s\\\\s\\\\\'~\\ “CC“wC»G.“MA“s1“CC“1C1“A““A“CC“CCAv.“A““CC“CCC‘“‘\»»\“A“M“A“Ax““xx“«\“CCC‘C‘“1C“ACC““WA1M1““CACCACG‘CCCGCCACCGCCC FPO/C 101—31151" 3;u,ununflymwuuu, C‘ *C‘*A ”.n”,,/,,(,,,,fl,”«mum, ,E3 , CTWTTPATGTGGTCT 1,- .

ACGAGGGTGGG1.C.AGG<.1..A1:G1.131AGC‘TThFC‘ 313TGGTG1‘AG'ATGAACTTCLAGGGTCA (“mam "EGG" GCATSG'V'CC"fiflfiwm 333333333333333333333333333333333333333333333333333333333333333 I A*A*G*TUGTGCGC BFPONC 201met T1CTCGACGT/ACCCTTCCGG CGACTTG.AAGA.111T1GTG: TGCT'1‘CA1T3TGG1‘CGG ‘CT-AL\CLU AAGCACTGCACGCCG.GCCTGAAGCITGGT" CGAGGGlCGGCCAGGGCACC1C1€HJAC‘{‘1"1C)(‘(Y‘3"”C‘rC‘rlC1CAGATGAACTCAGGGlCA51:0 CTTGCCG.1A1{31'(3131‘A{1:131:13 ‘V ‘ 3 ‘ ‘ 1 :- 1'11/{32-5‘1-3'1‘131' 1/4;14;I:A’1’;1);}!lII)4I!!!II!lI’ll/4’1/4;;a;ll;ll;lA:/A)IA I””atuunuaaflnun’ C'ICIAAUG 1 UCCCC‘.1C‘rCC‘C'1‘(1C;1“CC.AC.CC’ 1 C1"1'C'rAC‘.CACCI1C‘ACC:‘"ACC‘TCECG’TCIC‘AC; UC‘ '1"I‘C‘..L‘AC‘1(;C‘.C‘1C"1ACCCCTGAC‘C‘AC‘A'i'GAACTCAGC‘AC‘U"'"AC"1”1IC‘.1"1'CTAACT'1‘C‘CGCCA'1'C‘xCTCCGA AAGCTG(\(‘CCl'GAA'C" “TCA'1C1GC ‘ * GC.‘’XAGCTGCL.l'.”Cl-TGCCCTU-CF‘SACCCTACTGAC'C'AC‘C‘TTCACCC‘Af‘i3111GTGC.AGTGC. '1TCAGCCGC'IACCCCGACC‘AC‘A1C)AAGC‘AGCACGAC‘TC'1TCAAG'1CCGCCATGC.(ICGA AGGC'l'ACGT-‘CCAGC-A GC- " 1'*C‘* . “T ' A“CC“1““w“1"1w“wC»C~C1~‘3“mCACCACAA“AC“““Cw.““‘“~n~“»““~-“~»“»ww AA“ ““‘waCanxxwanw“1C3~C»C3~G»~~A“ACCC“CC““CCCCCCCCCACAW.1“““»»““\“““\““ = P8 linkage. (phosphothioate) 11311223: {21111313131131} Taiee ageing“13(33'3/3’11131‘1ahe1edGRONS [11132.14] 11113. purpose of This series of experiments is To compare the encies of phoephothioate (PS) fabeled GRONS (having 3 PS 1110113111253 at each end of The GRON) To the S’Cyffil' 331117 d GRONS. ’Cy'fi/3’111C1abeledGRO’Ns have a 5* Cy?) fluei‘ephm‘e (amidiie) and a 3’ 111C reverse base. Efficiency I213. assessed using eenveisien 131' 131116 fluerescent protein (BF?) to green fluorescence {1111215} In 311 three experiments, done either by PEG delivery of GRONS into protoplasts in individual 0011 Tubes (labeled ‘”1’ubes”) 01111 96-well plates (1abe1ed “'96— 331211111811"), there was no significant. difference betw $811 the different GRON chemistries in B1313 10 C1131? conversion efficiency 1-113 determined by cyternetry (171g. 1) . e 3: Cempariseii between the 4141113? BFPMNC §’-3PS/ 3’~3PS GRON and flicazaki Fragment GRUNS [802161 The purpose of This series 01' expefimems is to compare the. conversion efficiencies of the. phosp‘hothioate (PS) labeled GRONS with BPS n'ioieties at each end of SUBSTITUTE SHEET (RULE 26) the GRON t0 “Okazaki fragment GRONs” in the presence and absence of a member of the bleomycin family, ZeocinTM (1 mg/ml) to induce DNA breaks. The design of these GRONs are depicted in Fig. 2. GRONs were delivered into Arabidopsis BFP protoplasts by PEG treatment and BFP to GFP conversion was determined at 24 h post treatment by cytometry. Samples treated with zeocin (1 mg/ml) were incubated with zeocin for 90 min on ice prior to PEG treatment.

In general the presence of zeocin (1 mg/ml) increased BEP to GFP conversion as determined by try (Table 2). In both the presence and absence of zeocin, the NC Okazaki GRON containing one 2’-O Me group on the first RNA base at the 5’ end of the GRON was more cious at converting BFP to GFP when compared to the NC Okazaki GRON containing one 2’—O Me group on each of the first nine 5’ RNA bases (Fig. 2 and Table 2).

In all experiments, there was no significant difference between the 41—mer BFP4/NC 5’3PS/ 3 ’3PS and the 7 l—mer Okazaki Fragment BFP4/NC GRON that contains one 5’ 2’—O me group on the first 5’ RNA base ed as BFP4 7l~mer (1) NC) in BFP to GFP conversion in both the ce or absence of 1 mg/ml of zeocin as determined by cytometry (Fig. 2 and Table 2). It is important to note that in the presence of zeocin (and expected for bleomycin, phleomycin, tallysomycin, pepleomycin and other members of this family of antibiotics) that conversion s strand independent (i.e., both C and NC GRONS with the designs tested in these ments display imately equal activity). 2014/029566 £08219] Table 2: Comparison of a standard GRDN design with Okazaki fragment GRON s in the presence and absence of a giyeopeptide antibiotic zeocin.

Zeocin (+) -01.2275. 2m_355. -::_.3.2 . 1. . . x ’7 =-‘ . _.L 12949 ' ’ ’ 004879 . 0.0010(3i (21.034503 . 1 ". I. . , ’10JO75 Examgie 4: Comparison between the 41-min} 1911111151 and Ziii-mer BFPMNC S" 3335! 3’-3§’S GRQNS The purpose 0111115; series of experim 6.1115- was. to compexe the conversion efficiencies {in the. presence and absence of zeocin) of the phosplrlotiiioaie (PS) iabe ed GRGNS with 398 moieties at each end. of the GRON of ent lengths: 414116.13 10L mer and 201—mer51111111111111 Table 1. Again, the presence of zeocin {1 ) increased BF? {0 GP? (3011176131011 17311.“ as dete1111i11ed bv eytomeuy (iabie 3) Ike Ovemli [1131111111 ail three nenis was linear with increasing NC GRON length in 110111 the. presence. and absence. of 261313.111 Except for the EFF-LVN 3/101 and C/101 in the presence of it(19111 this bad conversion rates t‘hat wen: ‘iosc to equai but lowm 1113111116: 11me:NC GRON. This is in contrast to 2131 previous experiments in which [he EFF—4M} coding and SUBSTITUTE SHEET (RULE 26) PCT/U82014/029566 mmcading GRONS WerC. used Whmein the non-Coding was always far supeiior m the coding GRON Ibis n‘y in sion frcquency also applies to {he EFF-4&0} GRONS used in this cxpen’mcntai scrim. {@9221} Tahit: 3: Exp. Brmnmex- .1 2 :8 242011113 Naznc ’ :22222 ; 222222- 222222222222 22272M2222 2222 — Stz'DLv$83095 0‘9"” ! C:.12‘§9t79 Pfixbflé 9—29063 22222 i (10417103 00?'88424 Of‘1‘“,‘26 0”0222390 (100/0711 0.02930445 00015002 O.0557584 0.01 J0017 00037505 0005000)) [86222 Example 5: S combined with GRONs to impmve conversion in plants. {00223} Times design con‘xponents must be. considered when assembling a CRESPR cumplex: C2359, gRNA (guide. RNA) and {hrs target region (prom—spacer in endogenaus target gene).

SUBSTITUTE SHEET (RULE 26) Gas 9 - Transient expression of Cas9 gene from Streptococcus pyogenes codon optimized for Arabidopsis or corn driven by 358 or corn ubiquidn respectively. Optimized genes synthesized by Genewiz or DNA 2.0. NB must ensure no cryptic introns are created.

~ RBCSE9 terminator as per Gl 155 - Single SV40 NLS (PKKRKV) as a C-terminal fusion ~ The vector backbone would be as per ail our transient expression systems — G1 155. gRNA - pre—creRNA as per Le Cong et al., 2013 and - Propose to use a chimeric trachNA Jinek et al., 2013. Note that LeCong et al. showed that the native full length tracr + pre—chNA complex cleaved much more efficiently than the ic version. An option therefore would be to make a chimera using the full length (89bp) trachNA. - ce of gRNA ( (N)20 represents guide sequence). The bracketed sequence comprises the full length 89bp form.

NNNNNNNNNNNNNNNNNNNN(i1“l"!"¥‘1\GA(TCTAGAA9‘1"!“thTAAG’FYAAAATA ’1‘A(?'1‘("C€j[Tl‘A'l’G’fl'CTE’GAAAAA (\{E'E‘GM3T0{:E‘CACflKMG'YCG G’E’GG'I‘G (11‘1"?! ”l ”'1 '1 Figure 3 uced from Cong et al., shows the native x and the chimera.

[Text continued on page 64] PCT/U82014/029566 - The gRN'A would be expressed under the ALUS RNA pol Ill promoter in zlmbidapsis (sequence given below). 111 com the 21111.76 RNA pol HI promoter could be used.

These choices are based cm, Wang (a: at. 2008.

— RBCSEQ terminatm' as per 81155 at a string 01”le as per Wang er: a]. 1.2013. and the one—component approach shown belew.

At U6 pmmoter sequence from Wang et at {69227} ’l‘argetregiam — The guide sequence specificity is defined by the target region sequence, lnrespeetlve of the choice of model Ot‘ganisn't this will be the Y66H locus of BFP. A PAM (NGG) sequence in the Vlclnity of Y66H is the only design t‘estrietien. Alsu, including the Ytfitilrl position in the 3’ lilbp of the guide sequence (“seed sequence”) would mean that. once repair has been achieved the site will not get re«cutt.

Te gtg ace ace tie ace. cac ggc VTTFTY 6} 62 63 64 65 66 6’7 {@9228} A ct vector backbone from (31155 will be needed in order to enable 00— delivery of Cast) and gRNA. This m will be ‘wented with the one—cemptment approach: {062233} One cannpenent approacl't Will-336} Le Cong et at. (2013:) used a simplified approach, expressing both the gRNA and the (38.59 as a single transient constmct. driven by the pol Ill {36 prom-bier, as nutlined below. In this way, for a given crop, multiple genes could be targeted by simply swapping in the guide insert ce. We. would replace the EFlu pmmoter for one le. for the crop (pMAS for At, Ubi far . For the terminator we would use RBCSEQ. The NLS used in plants would be a single th’e‘cminal 5V5“) as outlined above.

SUBSTITUTE SHEET (RULE 26) Note that in the construct below a truncated gRNA is used Where the tracer RNA region is not included. The authors showed that in humans that this was less effective at guiding the Cas9 that the full length version. It is therefore proposed that the full length gRNA to be used here. Notably in a subsequent paper using CRISPRs in yeast, DiCarlo et al. (2013) used the full length version. The te would be cloned into a G1155 background.

Figur 4 shows a schematic of the expression vector for chimeric chNA. The guide sequence can be inserted between two BbsI sites using annealed oligonucleotides. The vector already contains the partial direct repeat (gray) and partial trachNA (red) ces. WPRE, uck hepatitis virus post transcriptional regulatory element.

In vivo assay Transient option - One approach to confirm target recognition and nuclease activity in planta would be to emulate the YFP single stranded annealing assay which Zhang et al. (2013) used for . The spacer sequence (target sequence) plus PAM would need to be inserted into the YFP or lent gene.

— Transient option - The TALEN — BFP system could be used as a control.

- Whilst the above approach would be an on—going tool for confirming functionality of a given CRISPR system for a given spacer sequence, proof of t of the activity of CRISPRs in plants would be to use the GFP system.

[Text continued on page 66] PCTfUSZOl4/029566 - Here the s used for BFPHGFP could be co—transformed into At together with G1155 and no GRON. If cutting were efficient enough, a ion in GFP expression could be apparent. This would likely require optimization of plasmid loading.

- Once activity is confirmed a genomic BFP target would be targeted with a Visual and sequence~based ut.

In vitro assay - In order to y confirm activity of a CRISPR system, an in vitro assay could be used as per Jinek et al 2012. Here a pie—made and purified S.pyogenes Cas9 is incubated with synthesized gRNA and a plasmid containing the recognition sequence. sful cleavage is analysed by gel electrophoresis to look for cut plasmids.

Detailed protocol: Plasmid DNA cleavage assay. Synthetic or in vino-transcribed trachNA and chNA were pre—annealed prior to the reaction by heating to 95°C and slowly cooling down to room ature. Native or restriction digest—linearized plasmid DNA (300 ng (~8 nM)) was incubated for 60 min at 37°C with punfied Cas9 protein (50-500 nM) and trachNA:chNA duplex (50—500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or t 10 mM MgC12. The reactions were stopped with 5X DNA loading buffer containing 250 mM EDTA, resolved by 0.8 or 1% agarose gel electrophoresis and visualized by um bromide staining. For the Cas9 mutant cleavage assays, the reactions were stopped with 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior to loading on the agarose gel.

Trait targets in Crops Given the lity of the CRISPR recognition ce it is not difficult to find potential protospacer sequences as defined by a 3’ NGG PAM sequence.

ZmEPSPS The example below shows a suitable protospacer sequence (yellow) and PAM (blue) in order to create a DS break in the catalytic site of ZmEPSPS where mutations at PCT/USZOI4/029566 the T97 and P101 are known to cause glyphosate tolerance. Subsequent oligo-mediated repair (ODM) of the break would result in the desired changes.

TAMRPLTVAAV act gca atg cgg cca ttg The table below gives the protospacer sequences of genes of st in crops of interest: S: 9.101. écggctgcagttactgctgct. 5.

EEPSPS 2'25 P101 ge"?9:59:59--.-........,...§ 1.918.993???9.969%??? ............

EEPSPS P101 38989599998993"?............ ’ "" i'é'ééiéiéiEiéliZi-iélélé51" 3899?.9..... ‘33 CESFFSEFEQSGE E§?.§EEE‘E13.19.11,..... gcgcctcgct PPXAZZOattttacaggtgtttacgcc .....

A limitation of the design constraints is that it is often hard to find a NGG sequence within 12 bp of the nucleotide being altered by ODM. This is significant because if this was the case, successful ODM would mean that subsequent g would not be possible because the protospacer seed sequence would be altered. Jinek et al. (2012) showed this was detrimental to cutting efficiency. nces LeCong eta12013 Science : vol. 339 no. 6121 pp. 819—823.

Jinek er a] 2012 Science. 3371816~21 Wang et al 2008 RNA 14: 903—913 Zhang eta12013. Plant Physiol. 161: 20~27 One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are ary, and are not intended as tions on the scope of the invention.

It will be readily nt to a person skilled in the art that varying substitutions and modifications may be made to the invention sed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the ication are indicative of the levels of those of ordinary skill in the art to which the invention ns. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically sed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been ed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of ing any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be ed to by those skilled in the art, and that such modifications and variations are ered to be within the scope of this invention as defined by the appended claims.

] Other embodiments are set forth within the following claims.

Claims (8)

1. Amethod for introducing a gene repair oligonucleobase (GRON-mediated mutation into a target deoxyribonucleic acid (DNA) sequence in a plant cell, comprising: delivery ofa GRON and a site-specific se selected from the group consisting ofa zinc finger nuclease, a Transcription Activator—Like Eifector Nuclease (TALEN) and a Clustered Regularly paced Short Palindromic Repeat complex (CRISPR complex) into the plant cell, wherein the GRON hybridizes at the target DNA ce to create a mismatched base—pair(s), which acts as a signal to attract the cell’s gene repair system to the site where the mismatched base—pair(s) is created, and is degraded after designated nucleotide(s) within the target DNA sequence is corrected by the cell's gene repair system such that the plant cell introduces the GRON-mediated mutation into the target DNA sequence and the plant cell is non—transgenic ing the introduction; whereinthe GRON comprises one ormore alterations from conventional RNAandDNAnucleotides at the 5' or 3' end thereofselected from a reverse base at the 3' end; one or more 2'O—methyl nucleotides at the 5’ or 3' end; one or more 2’-O-(2—methoxyethyl) nucleotides at the 5' or 3’ end; one or more phosphothioate modifications; a 5’ terminus cap; and one or more fluorescent dyes ntly attached thereto; and wherein the site-specific nuclease is designed to match the target DNA sequence.

2. The method of claim 1, n the GRON further comprises one or more of the following characteristics; the GRON is greater than 55 bases in , the GRON comprising two or more mutation sites for introduction into the target DNA; the GRON ses one or more a basic nucleotides; the GRON comprises one or more 8'oxo dA and/or 8‘oxo dG nucleotides; the GRON comprises one or more 2'0—methyl RNA nucleotides at the 5’ end f; the GRON comprises at least two 2'—O—methyl RNA nucleotides at the 5’ end f; the GRON comprises an intercalating dye; the GRON comprises a backbone modification selected fi‘om the group consisting of a methyl phosphonate modification, a locked nucleic acid (LNA) modification, 21 O -(2—methoxyethyl) (MOE) modification, a di PS modification, and a peptide nucleic acid (PNA) ation; the GRON comprises one or more intrastrand crosslinks; and the GRON comprises one or more bases which increase hybridization energy.

3. The method ofclaim 1 or 2, wherein the method further comprises synthesizing all or a portion of the GRON using nucleotide multimers.

4. The method ofany one of claims 1 — 3, wherein the target deoxyribonucleic acid (DNA) sequence is within the plant cell nuclear genome, the chloroplast genome or the mitochondrial genome.

5. The method of any one of claims 1-4, wherein the plant cell is a species selected fiom the group consisting ofcanola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfafa, m, tomato, mango, peach, apple, pear, strawbeny, banana, melon, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, fababean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans, , pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turfand forage grasses, flax, oilseed rape, d, cucumber, g glory, , pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plant.

6. The method ofany one ofclaims 1—5, n the target DNA sequence is an endogenous gene of the plant cell.

7. The method ofany one ofclaims 1—6, further comprising regenerating a plant from the plant cell.

8. The method ofclaim 7, r comprising collecting seeds fi'om the plant.