BR112016021092B1 - METHOD FOR CAUSING A GENETIC ALTERATION IN A PLANT CELL - Google Patents

Abstract

METHODS AND COMPOSITIONS FOR INCREASING THE EFFICIENCY OF TARGETED GENE MODIFICATION USING OLIGONUCLEOTIDE-MEDIATED GENE REPAIR. Provided herein are methods and compositions for making targeted changes to a DNA sequence. In various aspects and embodiments, methods and compositions are provided for modifying a DNA sequence in a cell (such as a plant, bacterial, yeast, fungal, algal, or mammalian cell). In some aspects and embodiments, DNA modification involves combining gene repair oligonucleotides with approaches that increase the availability of components of the target cell's gene repair mechanisms, such as a DNA cutter.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of US Provisional Application No. 61/953,333, filed on March 14, 2014; US Provisional Application No. 62/051,579, filed on September 17, 2014; US Provisional Application No. 62/075,811, filed on November 5, 2014; 62/075,816, filed on November 5, 2014; and US Provisional Application No. 62/133,129, filed March 13, 2015, each of which is incorporated herein by reference in its entirety, including all tables, figures and embodiments.

FIELD OF INVENTION

[0002] The present description relates, at least in part, to selective genetic mutations and modifications, including methods and compositions for carrying out such mutations and modifications.

FUNDAMENTALS

[0003] The following discussion is merely provided to aid the reader in understanding and is not intended to describe or constitute the prior art of the present disclosure.

[0004] US Patent No. 6,271,360 discloses methods and compositions for introducing genetic modifications into predetermined target genes of a living cell by introducing an oligodeoxynucleotide that encodes the predetermined change. Oligodeoxynucleotides are effective in bacterial, mammalian, avian and plant cells.

[0005] US Patent No. 8,771,945 discloses vectors and vector systems, some of which encode one or more components of a CRISPRS complex, as well as methods for creating and using such vectors.

[0006] US Patent No. 8,470,973 "refers to methods for selectively recognizing a base pair in a DNA sequence of a polypeptide, modified polypeptides that specifically recognize one or more base pairs in a DNA to DNA sequence that is modified so that it can be specifically recognized by a polypeptide and the uses of the polypeptide and DNA in targeting specific DNA, as well as methods of modulating the expression of target genes in a cell."

SUMMARY

[0007] Provided herein are methods and compositions for effecting a targeted genetic alteration of DNA in a cell. Certain aspects and modalities relate to improving the efficiency of targeting modifications to specific sites in genomic or other nucleotide sequences. As described herein, nucleic acids that direct specific changes to the genome can be combined with various approaches to increase the availability of components of natural repair systems present in the cells being targeted for modification.

[0008] In a first aspect, methods are provided for introducing an oligonucleobase-mediated mutation repair gene (GRON) into a target deoxyribonucleic acid (DNA) sequence in a plant cell. In certain embodiments the methods may include, inter alia, culturing the plant cell, under conditions that enhance one or more cellular DNA repair processes, prior to and/or coincident with the delivery of a GRON into the plant cell; and/or delivering a GRON into the plant cell greater than 15 bases in length, a GRON optionally comprising one or more; or two or more; mutation sites for the introduction of target DNA.

[0009] An "oligonucleotide repair gene" or "GRON", as used herein, means an oligonucleobase (e.g., mixed duplex oligonucleotides, molecules containing non-nucleotides, single-stranded oligodeoxynucleotides, double-stranded oligodeoxynucleotides, and other molecules gene repair), which may, under certain conditions, direct single, or in some embodiments multiple, nucleotide deletions, insertions or substitutions in the DNA sequence. This oligonucleotide-mediated gene repair editing of the genome can comprise both non-homology-based repair systems (e.g., non-homologous end joining) and homology-based repair systems (e.g., homology-directed repair). GRON is normally intended to align in register with a genomic target, except for engineered mismatch. These mismatches can be recognized and corrected through the mobilization of one or more of the cell's endogenous DNA repair systems. In some embodiments a GRON or oligonucleotide may be designed to contain several differences when compared to the sequence of target organisms. These differences may not all affect the protein sequence translated from said target sequence and in one or more cases be known as silent changes. Numerous variations of GRON structure, chemistry, and function are described in this document. In various embodiments, a GRON as used herein may have one or more modifications. For example, a GRON as used herein may have one or more modifications that attract DNA repair machinery to the target site (mismatch) and/or that prevent recombination of part or all of the GRON (with the exception of systematic elimination). desired, insertion(s), substitution(s) or the like) in the genomic DNA of the target DNA sequence and/or that increase the stability of GRON.

[0010] In various embodiments, a GRON may have both RNA and DNA nucleotides and/or other types of nucleobases. In some embodiments, one or more of the DNA or RNA nucleotides comprises a modification.

[0011] In one aspect, a method of causing a genetic change in a plant cell is provided, wherein the method involves exposing the cell to a DNA cutter and a GRON, e.g., a GRON that is modified, such as contemplated here. In some embodiments the GRON may be modified, such as with a Cy3 group, 3PS group, a 2'O-methyl group or other modification, as contemplated herein. In another aspect, a plant cell is provided that includes a DNA cutter and a GRON, for example, where the GRON is modified, such as with a Cy3 group, 3PS group, a 2'O-methyl group or other modification. In some embodiments, the DNA cutter is one or more selected from a CRISPRS, a TALEN, a zinc finger, meganuclease, and a DNA-cleaving antibiotic. In some embodiments, the DNA cutter is a CRISPRS. In some embodiments, the DNA cutter is a TALEN. In some embodiments, the GRON is between 15 and 60 nucleobases in length; or between 30 and 40 nucleobases in length; or between 35 and 45 nucleobases in length; or between 20 and 70 nucleobases in length; or between 20 and 200 nucleobases in length; or between 30 and 180 nucleobases in length; or between 50 and 160 nucleobases in length; or between 70 and 150 nucleobases in length; or between 70 and 210 nucleobases in length; or between 80 and 120 nucleobases in length; or between 90 and 110 nucleobases in length; or between 95 and 105 nucleobases in length; or between 80 and 300 nucleobases in length; or between 90 and 250 nucleobases in length; or between 100 and 150 nucleobases in length; or between 100 and 200 nucleobases in length; or between 100 and 210 nucleobases in length; or between 100 and 300 nucleobases in length; or between 150 and 200 nucleobases in length; or between 200 and 300 nucleobases in length; or between 250 and 350 nucleobases in length; or between 50 and 110 nucleobases in length; or between 50 and 200 nucleobases in length; or between 150 and 210 nucleobases in length; or between 20 and 1000 nucleobases in length; or between 100 and 1000 nucleobases in length; or between 200 and 1000 nucleobases in length; or between 300 and 1000 nucleobases in length; or between 400 and 1000 nucleobases in length; or between 500 and 1000 nucleobases in length; or between 600 and 1000 nucleobases in length; or between 700 and 1000 nucleobases in length; or between 800 and 1000 nucleobases in length; or between 900 and 1000 nucleobases in length; or between 300 and 800 nucleobases in length; or between 400 and 600 nucleobases in length; or between 500 and 700 nucleobases in length; or between 600 and 800 nucleobases in length; or more than 30 nucleobases in length; or more than 35 nucleobases in length; or more than 40 nucleobases in length; or more than 50 nucleobases in length; or more than 60 nucleobases in length; or more than 65 nucleobases in length; or more than 70 nucleobases in length; or more than 75 nucleobases in length; or more than 80 nucleobases in length; or more than 85 nucleobases in length; or more than 90 nucleobases in length; or more than 95 nucleobases in length; or more than 100 nucleobases in length; or more than 110 nucleobases in length; or more than 125 nucleobases in length; or more than 150 nucleobases in length; or more than 165 nucleobases in length; or more than 175 nucleobases in length; or more than 200 nucleobases in length; or more than 250 nucleobases in length; or more than 300 nucleobases in length; or more than 350 nucleobases in length; or more than 400 nucleobases in length; or more than 450 nucleobases in length; or more than 500 nucleobases in length; or more than 550 nucleobases in length; or more than 600 nucleobases in length; or more than 700 nucleobases in length; or more than 800 nucleobases in length; or more than 900 nucleobases in length.

[0012] GRONs can be targets of both the non-coding (NC) and coding (C) regions of a target gene. By way of example, Figs. 27 and 28 show, respectively, C-GRONs and NC-GRONs suitable for introducing mutations into the rice genome in order to introduce one or more of the following amino acid substitutions for the ACCase gene. The convention is to use the amino acid numbering system for foxtail plastid ACCase (Alopecurus myosuroides; Am) as the reference. The ACCase numbering used herein is based on the numbering for the foxtail ACCase protein reference sequence (SEQ ID NO: 1) or an analogous amino acid residue in an ACCase paralog (V=CY3; H= 3'DMT dC CPG). The following table lists ACCase mutations that produce one or more of aloxidim, butroxidim, clethodim, cloproxidim, cycloxidime, setoxidim, tepraloxidime, tralkoxidime, clorazifop, clodinafop, clofop, diclofop, fenoxaprop, fenoxaprop-P, fentiaprop, fluazifop, fluazifop-P , haloxifop, haloxifop-P, isoxapirifop, propaquizafop, quizalofop, quizalofop-P, trifop, pinoxaden, salts and esters of any of these agronomically acceptable herbicides, and combinations thereof with a resistant phenotype.

[0013] Similarly, Figs. 29 and 30 respectively represent C-GRONs (coding) and NC-GRONs (non-coding) suitable for introducing mutations into the flax genome to introduce one or more of the following amino acid substitutions for the EPSPS gene (with all numbering relative to the amino acid sequence of the E. coli AroA protein (prokaryotic equivalent of EPSPS) (as described in US Patent No. 8268622 (V=CY3; H=3'DMT dC CPG). EPSPS that produce glyphosate and agronomically acceptable ester salts of any of these herbicides, and resistant phenotype combinations thereof.

[0014] The term "CRISPR" as used herein refers to elements; that is, a cas gene (associated with CRISPR), transcript (e.g. mRNA) or protein and at least one CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats, also known as SPIDRs - SPacer Interspersed Direct Repeats) sequence; which, when actually present or expressed in a cell can effect the cleavage of a target DNA sequence through the CRISPRS/CAS cellular machinery as described in, for example, Cong, L. et al., Science, vol. 339 no 6121 pp. 819-823 (2013); Jinek et al, Science, vol. 337:816-821 (2013); Wang et al., RNA, vol. 14, pp. 903-913 (2008); Zhang et al., Plant Physiology, vol. 161, pp. 20-27 (2013), Zhang et al, PCT Application No. PCT/US2013/074743; and Charpentier et al., PCT Application No. PCT/US2013/032589. In some embodiments, such as, for example, a CRISPR for use in a eukaryotic cell, a CRISPR as contemplated herein may also include a supplementary element that includes a sequence of one or more functional nuclear localization signals. CRISPRs, as contemplated herein, may be expressed in, administered to, and/or present in a cell (such as a plant cell) in any of many forms or manifestations. For example, a CRISPR as contemplated herein may include or involve one or more of a CRISPR on a plasmid, a CRISPR nickase on a plasmid, a CRISPRSa on a plasmid, or a CRISPRi on a plasmid as follows:

[0015] CRISPR on a plasmid: A recombinant expression vector comprising: (i) a nucleotide sequence encoding a target RNA-DNA (e.g., targeting RNA), wherein the RNA-targeting DNA comprises: a. a first segment comprising a nucleotide sequence that is complementary to a target DNA sequence (e.g., protospacer, spacer or crRNA); and b. a second segment that interacts with a polypeptide directed to the modification site (e.g., trans-activation crRNA or tracrRNA); and (ii) a nucleotide sequence encoding the modification site-directed polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises: a. an RNA-binding moiety that interacts with target RNA-DNA (e.g., REC lobe); and b. a portion of activity that causes double-strand breaks within the target DNA (e.g., NUC lobe), where the site of the double-strand breaks in the target DNA is determined by DNA-targeting RNA.

[0016] CRISPR nickase on a plasmid. A recombinant expression vector comprising: (i) a nucleotide sequence encoding a target RNA-DNA (e.g., targeting RNA), wherein the RNA-targeting DNA comprises: a. a first segment comprising a nucleotide sequence that is complementary to a target DNA sequence (e.g., protospacer, spacer or crRNA); and b. a second segment that interacts with a polypeptide directed to the modification site (e.g., trans-activation crRNA or tracrRNA); and (ii) a nucleotide sequence encoding the modification site-directed polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises: a. an RNA-binding moiety that interacts with target RNA-DNA (e.g., REC lobe); and b. a portion of activity that causes double-strand breaks within the target DNA (e.g., NUC lobe), where the site of the double-strand breaks in the target DNA is determined by DNA-targeting RNA.

[0017] CRISPRa on a plasmid. A recombinant expression vector comprising: (i) a nucleotide sequence encoding a target RNA-DNA (e.g., targeting RNA), wherein the RNA-targeting DNA comprises: a. a first segment comprising a nucleotide sequence that is complementary to a target DNA sequence (e.g., protospacer, spacer or crRNA); and b. a second segment that interacts with a polypeptide directed to the modification site (e.g., trans-activation crRNA or tracrRNA); and (ii) a nucleotide sequence encoding the modification site-directed polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises: a. an RNA-binding moiety that interacts with target RNA-DNA (e.g., REC lobe); and b. a portion that modulates transcriptional activity (e.g., NUC lobe; in certain embodiments increases transcription) within the target DNA, wherein the site of transcriptional modulation within the target DNA is determined by the DNA-targeting RNA.

[0018] CRISPRi on a plasmid. A recombinant expression vector comprising: (i) a nucleotide sequence encoding a target RNA-DNA (e.g., targeting RNA), wherein the RNA-targeting DNA comprises: a. a first segment comprising a nucleotide sequence that is complementary to a target DNA sequence (e.g., protospacer, spacer or crRNA); and b. a second segment that interacts with a polypeptide directed to the modification site (e.g., trans-activation crRNA or tracrRNA); and (ii) a nucleotide sequence encoding the modification site-directed polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises: a. an RNA-binding moiety that interacts with target RNA-DNA (e.g., REC lobe); and b. a portion that modulates transcription/translation activity (e.g., NUC lobe; in some embodiments decreases transcription/translation) within the target DNA, wherein the site of transcription/translation modulation within the target DNA is determined by the targeted RNA to DNA.

[0019] Each of CRISPR on a plasmid, CRISPR nickase on a plasmid, CRISPRa on a plasmid, and CRISPRi on a plasmid may, in some alternative embodiments, have administered one or more appropriate elements, expressed or present in a cell as an RNA (e.g., mRNA) or a protein, rather than on a plasmid. Delivery of mRNA can be protected as described in Kariko et al, US Patent No. 8,278,036.

[0020] In some embodiments, each of CRISPRi and CRISPRa may include a deactivated Cas9 (dCas9). A deactivated Cas9 still binds to target DNA but has no cutting activity. Deficient Nuclease-Cas9 may result from point mutations D10A and H840A that inactivate its two catalytic domains.

[0021] In some embodiments, a CRISPRi inhibits transcription initiation or elongation through steric hindrance of RNA polymerase II. CRISPRi can optionally be enhanced (CRISPRei) by fusing a strong repressor domain to the C-terminal end of a dCas9 protein. In some embodiments, a repressive domain recruits and employs chromatin modifiers. In some embodiments, the repressor domain may include, but is not limited to, domains as described in Kagale, S. et al., Epigenetics, vol. 6 no 2 pp141-146 (2011): 1. LDLNRPPPVEN - OsERF3 repressor domain (LxLxPP, motif) 2. LRLFGVNM - AtBRD repressor domain (R/KLFGV, motif) 3. LKLFGVWL - AtHsfB1 repressor domain (R/KLFGV, motif) 4. LDLELRLGFA - AtSUP repressor domain (EAR motif) IISLELEIGLINESEQDL DLELRLGFA*- complete AtSUP gene containing repressor domain (EAR motif)

[0022] In some embodiments, an activation of CRISPRa transcription is achieved through the use of dCas9 protein containing a fused C-terminal transcription activator. In some embodiments, an activation may include, but is not limited to, VP64 (VP16 4X), AtERF98 activation domain, or AtERF98x4 concatemers as described in Cheng, AW et al., Cell Research, pp1-9 (2013); Perez-Pinera, P. et al., Nature Methods, vol. 10 pp 913-976 (2013); Maeder, ML. et al., Nature Methods, vol. 10 pp 977-979 (2013) and Mali, P., et al., Nature Biotech., vol. 31pp 833-838 (2013).

[0023] In some embodiments CRISPR includes a nickase. In certain embodiments, two or more CRISPR nickases are used. In some embodiments, the two or more nickases are cleaved on opposite strands of target nucleic acid. In other embodiments, the two or more nickases cleave on the same target nucleic acid strand.

[0024] As used herein, "repressor protein" or "repressor" refers to a protein that binds to the DNA or RNA operator to prevent transcription or translation, respectively.

[0025] As used herein, "repression" refers to the inhibition of transcription or translation by binding of the repressor protein to the specific site on DNA or mRNA. In some embodiments, repression includes a significant change in the level of transcription or translation of at least 1.5-fold, in other embodiments at least two-fold, and in other embodiments at least five-fold.

[0026] As used herein, an "activator protein" or "activator" with respect to gene transcription and/or translation, refers to a protein that binds to the DNA or RNA operator to enhance or increase transcription or translation, respectively.

[0027] As used herein with respect to gene transcription and/or translation, "activation" with respect to gene transcription and/or translation, refers to improving or increasing transcription or translation by binding of activating protein to specific site on DNA or mRNA. In some embodiments, activation includes a significant change in the level of transcription or translation of at least 1.5-fold, in some embodiments at least two-fold, and in some embodiments at least five-fold.

[0028] In certain embodiments, conditions that enhance one or more cellular DNA repair processes may include one or more of: introduction of one or more sites for GRON or plant cell DNA that are targets for DNA repair. base excision, the introduction of one or more sites into GRON or plant cell DNA that are targets for nonhomologous end joining, the introduction of one or more sites into GRON or plant cell DNA that are targets for microhomology-mediated end joining, the introduction of one or more sites into GRON or plant cell DNA that are targets for homologous recombination, and the introduction of one or more sites into GRON or plant cell DNA that are targets to effect repair (e.g., base-excision repair (BER); homologous recombination repair (HR); mismatch repair (MMR); non-homologous end joining repair (NHEJ), which includes classical NHEJ and alternatively, and nucleotide excision repair (NER).

[0029] As described herein, GRONs for use in the present invention may include one or more of the following conventional RNA and DNA nucleotide changes: one or more non-basic nucleotides; one or more 8'oxo Da and/or 8'oxo dG nucleotides; a reverse base at the respective 3' end; one or more 2'O-methyl nucleotides; one or more RNA nucleotides; one or more RNA nucleotides at the 5' end thereof, and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; in which one or more of the RNA nucleotides can still be modified; one or more RNA nucleotides at the 3' end thereof, and, in some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; in which one or more of the RNA nucleotides can still be modified; one or more 2'O-methyl RNA nucleotides at the respective 5' end, and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; an intercalating dye; terminal cap at 5'; a structure modification selected from the group consisting of a phosphorothioate modification, a methylphosphonate modification, a locked nucleic acid (LNA) modification, an O -(2-methoxyethyl) (MOE) modification, a di PS, and a peptide nucleic acid modification (PNA); one or more intrastrand cross-links; one or more fluorescent dyes conjugated there, and in some embodiments at the 5' or 3' ends of the GRON; and one or more bases that increase the hybridization energy. This list is not intended to be limiting.

[0030] The term "wobble base" as used herein refers to a change of one or more nucleotide bases from a reference nucleotide sequence, wherein the change does not change the amino acid sequence encoded by the nucleotide relative to to the reference sequence.

[0031] The term "non-nucleotide" or "abasic nucleotide" as used in the present invention refers to any group or compound that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine. It may have substitutions for 2' or 3' H or OH, as described in the art and here.

[0032] As described herein, in certain embodiments, GRON quality and conversion efficiency can be improved by synthesizing all or a portion of the GRON using nucleotide multimers, such as dimers, trimers, tetramers, etc. improving its purity.

[0033] In certain embodiments, the target deoxyribonucleic acid (DNA) sequence is within a plant cell, for example, the target DNA sequence is in the genome of the plant cell. The plant cell may be non-transgenic or transgenic, and the target DNA sequence may be a transgene or an endogenous gene of the plant cell.

[0034] In certain embodiments, conditions that enhance one or more cellular DNA repair processes comprise the introduction of one or more compounds that induce single or double DNA strand breaks in the plant cell before, or coincident with, or after delivery of GRON to the plant cell. Exemplary compounds are described herein.

[0035] The methods and compositions described here are applicable to plants in general. By way of example only, one plant species may be selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat (including but not limited to Triticum spp., Triticum aestivum, Triticum durum Triticum timopheevii , Triticum monococcum, Triticum spelta, Triticum zhukovskyi and Triticum urartu and hybrids thereof), barley (including but not limited to Hordeum vulgare L., Hordeum comosum, Hordeum depressum, Hordeum intercedens, Hordeum jubatum, Hordeum marinum, Hordeum marinum, Hordeum parodii, Hordeum pusillum, Hordeum secalinum, and Hordeum spontaneum), rice (including but not limited to Oryza sativa subsp. indica, Oryza sativa subsp. japonica, Oryza sativa subsp. javanica, Oryza sativa subsp. glutinosa (glutinous rice), Oryza sativa of Aromatic group ( e.g. basmati), and Oryza sativa (floating rice group), alfalfa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, bean soybeans, soybeans spp, sugarcane, peas, chickpeas, peas, fava beans, lentils, turnips, parsnips, Brussels sprouts, lupins, cauliflower, kale, faveira, poplar, pine, eucalyptus, grapes, citrus, triticale, alfalfa, rye (including but not limited to Secale sylvestre, Secale strictum, Secale cereale, Secale vavilovii, Secale africanum, Secale ciliatoglume, Secale ancestore and Secale montanum), oats, grass (including but not limited to lawns including Zoysia japonica, Agrostris palustris, Poa pratensis, Poa annua, Digitaria sanguinalis, Cyperus rotundus, Kyllinga brevifolia, Cyperus amuricus, Erigeron canadensis, Hydrocotyle sibthorpioides, Kummerowia striata, Euphorbia humifusae Viola arvensis) and forage grasses, flax, rapeseed, cotton, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, nut-bearing plants insofar as they are not specifically already mentioned. These can also be applied in whole or in part to all other biological systems, including but not limited to bacteria, yeast, fungi, algae and mammalian cells and even their organelles (e.g. mitochondria and chloroplasts). In some embodiments, the organism or cell is of a species selected from the group consisting of Escherichia coli, Mycobacterium smegmatis, Baccillus subtilis, Chlorella, Bacillus thuringiensis, Saccharomyces cerevisiae, Yarrowia lipolytica, Chlamydamonas rhienhardtii, Pichia pastoris, Corynebacterium, Aspergillus nigere and Neurospora crassa. In some embodiments, the yeast is Yarrowia lypolitica. In other embodiments, the yeast is not Saccharomyces cerevisiae. In some embodiments, the plant cell or plant is from a species selected from the group consisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Glycine max, Amaranthus tuberculatus, Linum usitatissimum and Zea mays. Plant species may be selected from the group consisting of monocotyledonous plants of the grass family Poaceae. The family Poaceae can be divided into two main clades, the clade containing the subfamilies Bambusoideae, Ehrhartoideae, and Pooideae (the BEP clade) and the clade containing the subfamilies Panicoideae, Arundinoideae, Chloridoideae,centothecoideae, Micrairoideae, Aristidoideae, and Danthonioideae (the PACCMAD clade ). The subfamily Bambusoideae includes tribe Oryzeae. Plant species may concern plants from the BEP clade, in specific plants from the subfamilies Bambusoideae, Ehrhartoideae. The BET clade includes subfamilies Bambusoideae, Ehrhartoideae, and the group Triticodae and no other groups of subfamily Pooideae. BET crop plants are plants grown for food or forage that are members of the BET subclade, e.g. barley, maize, etc.

[0036] In certain embodiments, the methods further comprise regenerating a plant having a mutation introduced by GRON from the plant cell, and may comprise collecting seeds from the plant.

[0037] In related aspects, the present description relates to plant cells comprising a genomic modification introduced by a GRON in accordance with the methods described herein, a plant comprising a genomic modification introduced by a GRON in accordance with the methods described herein , or a seed comprising a genomic modification introduced by a GRON in accordance with the methods described herein; or progeny of a seed comprising a genomic modification introduced by a GRON in accordance with the methods described herein.

[0038] Other embodiments of the present description will be evident from the following detailed description, exemplary embodiments, and the embodiments.

BRIEF DESCRIPTION OF FIGURES

[0039] FIG. 1 describes the conversion of BFP to GFP mediated by phosphorothioate (PS) labeled with GRONs (3 fractions of PS at each end of the GRON) and GRONs labeled with 5'Cy3/3'idC.

[0040] FIG. 2 represents GRONs comprising RNA/DNA, referred to herein as "2'-O-methyl GRONs."

[0041] AFIG. 3 is a schematic view of the location in the bfp gene where the CRISPRS BFP5 targets.

[0042] FIG. 4 shows the results of the effect of CRISPRS introduced with either Cy3 or 3PS GRONs at various durations, on the percentage of BFP to GFP conversion in a BFP transgenic Arabidopsis thaliana model system.

[0043] FIG. 5 shows the results of the effect of CRISPRS introduced with 3PS GRONs at various durations, on the percentage of BFP to GFP conversion in a BFP transgenic Arabidopsis thaliana model system.

[0044] FIG. 6 is a schematic view of the GRON projections used in Example 9.

[0045] FIG. 7 shows the measurement of the average percentage of GFP-positive protoplasts from a BFP transgenic Arabidopsis thaliana model system, as determined by flow cytometry from 71-mer GRONs.

[0046] FIG. 8 shows the measurement of GFP-positive protoplasts from the BFP transgenic Arabidopsis thaliana model system, as determined by flow cytometry from 201-mer GRONs.

[0047] FIG. 9 shows the effect of CRISPRS introduced with coding and non-coding GRONs on the average percentage of GFP positive cells in a BFP transgenic Arabidopsis thaliana model system.

[0048] FIG. 10 is a schematic view of tethering a single-stranded GRON or double-stranded DNA to the CRISPRS/CAS complex.

[0049] FIG. 11 shows the results of the effect of CRISPRS and GRONs in mediating BFP to GFP conversion in a BFP transgenic Arabidopsis thaliana model system of spacers of different lengths.

[0050] FIG. 12 shows the results of the effect of CRISPRS and GRONs in mediating conversion of BFP to GFP in a BFP transgenic Arabidopsis thaliana model system of spacers that were encoded on a plasmid (gRNA plasmid) or used as an amplified fragment (gRNA amplicon ).

[0051] FIG. 13 shows the results of the effect of CRISPRS and GRONs in mediating BFP to GFP conversion in a BFP transgenic Arabidopsis thaliana model system of unmodified vs. modified GRONs. of 41-mer modified 3PS.

[0052] FIG. 14 shows next-generation sequencing results of 3- and 6-week-old Linum usitatissimum (Flax) microcalluses derived from PEG of shoot tip protoplasts treated with CRISPR plasmid at T = 0.

[0053] FIG. 15 shows next-generation sequencing results of 3- and 6-week-old Linum usitatissimum microcalluses derived from PEG of CRISPR plasmid-treated shoot tip protoplasts at T = 0.

[0054] CRISPR-cas nuclease activity and gene editing in A. thaliana protoplasts. 16A: Distribution of indels based on size as determined by deep sequencing in protoplasts treated with CRISPRS-cas (BC-1) plasmid 72 h after delivery. Indels represented 0.79% of total readings. 16B: BFP to GFP editing measured by the percentage of fluorescent GFP protoplasts identified by flow cytometry 72 h after delivery of plasmid (BC-1) and GRON (CG-6). Data depicted is not normalized for transfection efficiency. Error bars are s.e.m. (n = 9).

[0055] FIG. 17 shows gene editing of A. thaliana protoplasts using CRISPR-cas combined with GRONs of different lengths and chemical modifications. 17a: Comparison of 3PS and unmodified GRONs in gene editing from BFP to GFP as measured by flow cytometry at 72 hours after plasmid delivery (BC-1) and GRONs (CG-1) or (CG-2). 17b: Comparison of GRON BFP lengths for GFP gene editing as measured by flow cytometry at 72 hours after plasmid delivery (BC-2) and GRONs (CG-5) or (CG-8). 17c: Comparing GRONs from 3PS to 2'-O-Me for gene editing from BFP to GFP as measured by flow cytometry 72 h after plasmid delivery (BC-1) and GRONs (CG-6), (CG- 9) or (CG-10). 17d: Comparing GRONs from 3PS to Cy3 in gene editing from BFP to GFP as measured by flow cytometry 72 h after plasmid delivery (BC-3) and GRONs (CG-3) or (CG-4). Error bars are s.e.m. (n = 3). (CG-1): unmodified antisense BFP 41 nb; (CG-2): BFP antisense 41 nb 3PS modified; (CG-3): modified BFP sense 41 nb 3PS; (CG-4): modified BFP sense 41 nb Cy3; (CG-5): modified BFP sense 60 nb 3PS; (CG-6): BFP antisense 201 nb 3PS modified; (CG-8): modified BFP sense 201 nb 3PS; (CG-9): 201 nb 2'-O-Me antisense BFP modification at 5' first RNA base; (CG-10): 201 nb 2'-O-Me antisense BFP modifications on the first nine 5' RNA bases.

[0056] FIG. 18 shows TALEN nuclease activity and gene editing of A. thaliana and L. usitatissimum protoplasts. 18-A: Size-based distribution of indels as determined by deep sequencing in Arabidopsis protoplasts treated with TALEN (BT-1) plasmid at 72 hours post delivery. Indels represented 0.51% of total reads. 18b: BFP gene editing for GFP as measured by flow cytometry at 48 h after delivery of plasmid (BT-1) and GRON (CG-7). 18c: Representative distribution of indels based on bp length in L. usitatissimum protoplasts treated with a TALEN (LuET-1) targeting EPSPS genes 7 d after delivery. Total frequency of indels is 0.50%. d, L. usitatissimum EPSPS gene editing, as measured by deep sequencing at 7 d post delivery of plasmid (LuET-1) and GRON (CG-11) in protoplasts. Percentage of total reads represents the number of reads containing both P101A and T97I edits as a percentage of total reads. Error bars are s.e.m. (n = 3). (CG-7): modified BFP sense201 nb 3PS; (CG-11): EPSPS sense 144 nb Cy3 modified.

[0057] FIG. 19 shows effects of double-strand break inducing antibiotics zeocin and phleomycin on BFP editing for GFP in transgenic A. thaliana protoplasts. Protoplasts were treated with zeocin or phleomycin for 90 minutes before introduction of GRON PEG (CG2). Successful editing resulted in GFP-fluorescence. Green fluorescent protoplasts were quantified using an Attune Acoustic Focusing Cytometer.

[0058] FIG. 20 shows BFP transgenic A. thaliana cells converted five days after GRON delivery into transgenic BFP protoplasts, aiming for the conversion of BFP to GFP. Green fluorescence is indicative of BFP-GFP editing. A brightfield image; B, the same field of view in blue light. Error bars are s.e.m. (n= 4); (CG2): BFP antisense 41 nb 3PS modified; (CG12) Antisense BFP 41 nb 3PS modified non-targeting. Images were acquired with a Micro ImageXpress system (Molecular Devices, Sunnyvale, CA, USA), scale bar = 20 μm

[0059] FIG. 21 shows CRISPR-Cas and TALEN projections. 21A: Schematic representation of the CRISPR-Cas plasmid. The mannopine synthase (Mas) promoter is driving transcription of the Cas9 gene which is codon optimized for higher plants. The Cas9 gene contains two SV40 nuclear localization signals (NLS) at each end of the gene and a 2X FLAG epitope tag. The A. thaliana U6 promoter is directed to transcription from the gRNA scaffold and transcription is terminated using a poly(T) signal. 21B: Schematic representation of the TALEN plasmid. The Mas promoter is directed to transcription of the left and right arms of TALE linked together with a 2A ribosome skipping sequence. A Fok1 endonuclease is linked to the 3' end of each TALE arm. The 5' end of the left TALE contains a nuclear localization signal (NLS) and a V5 epitope tag. RBCT is the gene terminator of Pisum sativum RBCSE9.

[0060] FIG. 22 shows nuclease target regions of BFP and EPSPS. a, BFP target gene region for the CRISPR-Cas protospacers BC-1, BC-2 and BC-3 and TALEN BT-1, the left and right arms of TALE. The PAM sequence is shown in red. TALEN binding sites are in bold and underlined. The BFP editing site for GFP CAC^TAC (H66Y) is in bold green. b, EPSPS target gene region for TALEN, LuET-1, left and right arms of TALE. The EPSPS conversion site ACA> ATA and CCG> GCG (T97I and P101A) is in green.

[0061] FIG. 23 shows the amino acid sequence of the ACCase gene product of Alopecurus myosuroides (foxtail) (SEQ ID NO: 1).

[0062] FIG. 24 shows the amino acid sequence of the Escherichia coli EPSPS gene product (SEQ ID NO: 2).

[0063] FIG. 25 shows analogous positions of exemplary EPSPS.

[0064] FIG. 26 shows exemplary ACCase sequences.

DETAILED DESCRIPTION

[0065] Oligonucleotide-mediated targeted genetic modification is a valuable technique for use in specifically altering short stretches of DNA to create deletions, short insertions and point mutations. These methods involve DNA pairing/annealing, followed by a DNA repair/recombination event. First, the nucleic acid hybridizes with its complementary strand in double-stranded DNA in a process mediated by cellular protein factors. This annealing creates a centrally located mismatched base pair (in the case of a point mutation), resulting in a structural perturbation that most likely stimulates the endogenous protein machinery to initiate the second step in the repair process: site-specific modification of the chromosomal sequence. and/or in organelles (e.g., mitochondria and chloroplasts). This newly introduced mismatch induces the DNA repair machinery to execute a second repair event, which leads to the final proofreading of the target site. The present methods and compositions in various aspects and embodiments described herein can improve the methods by providing new approaches that increase the availability of DNA repair components, thereby increasing the efficacy and reproducibility of target nucleic acid gene repair-mediated modifications. .

[0066] Efficient methods for site-directed genomic modifications are desirable for research, clinical gene therapy, industrial microbiology and agriculture. One approach utilizes triplex-forming oligonucleotides (TFO) that bind as third strands to duplex DNA in a sequence-specific manner, to mediate site-directed mutagenesis. Such TFO can act either by delivery of a tethered mutagenic agent, such as psoralen or chlorambucil (Havre et al., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., J Virol 67 :7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995; Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991; Acids Res 25:3440-3444, 1997), or by binding with sufficient affinity to cause error-prone repair (Wang et al, Science 271: 802-805, 1996).

[0067] Another strategy for genomic modification involves the induction of homologous recombination between an exogenous DNA fragment and the target gene. This approach has been used successfully to target and disrupt selected genes in mammalian cells and has allowed the production of transgenic mice that carry knockouts of specific genes (Capeechi et al, Science 244: 1288-1292, 1989; Wagner, U.S. Pat. N ° 4873191). This approach involves the transfer of selectable markers to allow isolation of the desired recombinants. Without selection, the ratio of homologous to nonhomologous integration of transfected DNA in typical gene transfer experiments is low, generally in the range of 1:1000 or less (Sedivy et al, Gene Targeting, WH Freeman and Co., New York, 1992). This low efficiency of homologous integration limits the utility of gene transfer for experimental use or gene therapy. The frequency of homologous recombination can be enhanced by damage to the target site from UV irradiation and selected carcinogens (Wang et al, Mol Cell Biol. 8:196-202, 1988), as well as by site-specific endonucleases (Sedivy et al, Gene Targeting, W. H. Freeman and Co., New York, 1992; U.S.A. 92:806-810, 1995). Furthermore, DNA damage induced by triplex-targeted psoralen photoadducts can stimulate recombination within and between extrachromosomal vectors (Segal et al, Proc Natl Acad Sci, USA 92:806-810, 1995; Faruqi et al, Mol Cell Biol 16 : 6820-6828, 1996;

[0068] Linear donor fragments are more recombinogenic than their circular counterparts (Folger et al, Mol Cell Biol. 2: 1372-1387, 1982). Recombination can, in certain embodiments, also be influenced by the length of unbroken homology between both donor and target sites, with short fragments often appearing to be ineffective substrates for recombination (Rubnitz et al, Mol Cell Biol. 4:2253-2258 , 1984). However, the use of short DNA fragments or DNA/RNA hybrids for gene correction is the focus of several strategies. (Kunzelmann et al, Gene Ther 3:859-867, 1996).

[0069] "Nucleic acid sequence", "nucleotide sequence" and "polynucleotide sequence", as used herein refer to an oligonucleotide or a polynucleotide, and fragments or fractions thereof, and to DNA or RNA of genomic origin or synthetic which can be single or double stranded and represent the sense or antisense strand.

[0070] As used herein, the terms "oligonucleotides" and "oligomer" refer to a polymer of nucleobases of at least about 10 nucleobases and as many as about 1000 nucleobases.

[0071] The terms "DNA modification molecule" and "DNA modification reagent" as used herein refer to a molecule that is capable of recognizing and binding to a nucleic acid sequence in the genome of a cell. , specifically, and which is capable of modifying a target nucleotide sequence in the genome, wherein the recognition and specific binding of the DNA modification molecule with the nucleic acid sequence is protein independent. The term "protein-independent" as used herein in connection with a DNA-modifying molecule means that the DNA-modifying molecule does not require the presence and/or activity of a protein and/or enzyme for the recognition of and/or or specific binding to a nucleic acid sequence. DNA modification molecules are exemplified by, but not limited to, triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and those oligonucleotides that are intended to promote gene conversion. The DNA modifying molecules of the present disclosure are in certain embodiments distinguished from prior art nucleic acid sequences that are used for homologous recombination (Capecchi & Wong, Molec. Cell. Biol. 7: 22942295, 1987) in that the prior art nucleic acid sequences that are used for homologous recombination are protein dependent. The term as used herein in connection with a molecule "protein dependent" means that the molecule requires the presence and/or activity of a protein and/or enzyme for the recognition of and/or specific binding of the molecule to a sequence of nucleic acid. Methods for determining whether a DNA modifying molecule requires the presence and/or activity of a protein and/or enzyme for recognition of and/or specific binding to a nucleic acid sequence are within the skill in the art (see, for example, Dennis et al. Nucl. Acids Res. 27: 4734-4742, 1999). For example, the DNA modification molecule can be incubated in vitro with the nucleic acid sequence in the absence of any proteins and/or enzymes. Detection of binding between the DNA modification molecule and the specific nucleic acid sequence demonstrates that the DNA modification molecule is protein independent. On the other hand, the absence of binding between the DNA modification molecule and the specific nucleic acid sequence demonstrates that the DNA modification molecule is protein dependent and/or requires additional factors.

[0072] "Triplex-forming oligonucleotide" (TFO) is defined as a DNA or RNA sequence that is capable of binding in the major groove of a DNA or RNA duplex helix to form a triple helix. Although the TFO is not limited to any specific length, a preferred length of the TFO is 250 nucleotides or less, 200 nucleotides or less, or 100 nucleotides or less, or 5 to 50 nucleotides, or 10 to 25 nucleotides, or from 15 to 25 nucleotides. Although a degree of sequence specificity between the TFO and double-stranded DNA is necessary for triple helix formation, a certain degree of specificity is not necessary as far as the triple helix is capable of forming. Likewise, no specific degree of avidity or affinity between the TFO and the duplex helix is required as long as the triple helix is capable of forming. Although it is not intended to limit the length of the nucleotide sequence to which TFO specifically binds in one embodiment, the nucleotide sequence to which TFO specifically binds is 1 to 100, in some embodiments 5 to 50, in still other embodiments of 10 to 25, and in other embodiments from 15 to 25, nucleotides. Furthermore, "triple helix" is defined as a double helix nucleic acid with an oligonucleotide linked to a target sequence within the double helix nucleic acid. "Double-stranded" nucleic acid can be any double-stranded nucleic acid including double-stranded DNA, double-stranded RNA, and mixed duplexes of DNA and RNA. Double-stranded nucleic acid is not limited to any specific length. However, in preferred embodiments it is greater than 500 bp in length, in some embodiments greater than 1 kb and in some embodiments greater than about 5 kb. In many applications, double-stranded nucleic acid is cellular, genomic nucleic acid. The triplex-forming oligonucleotide can bind to the target sequence in a parallel or antiparallel manner.

[0073] "Peptide Nucleic Acids", "polyamides" or "PNA" are nucleic acids in which the phosphate structure is replaced by a polyamide structure based on N-aminoethylglycine. PNAs have a greater affinity for complementary nucleic acids than their natural counterparts, following the Watson-Crick base pairing rules. PNAs can form highly stable triple helix structures with DNA of the following stoichiometry: (PNA)2.DNA Although peptide and polyamide nucleic acids are not limited to any specific length, a preferred length of peptide and polyamide nucleic acids is 200 nucleotides or less, in some embodiments 100 nucleotides or less, and in some embodiments, 5 to 50 nucleotides in length. Although it is not intended to limit the length of the nucleotide sequence to which the peptide nucleic acid and polyamide specifically bind, in one embodiment, the nucleotide sequence to which the peptide nucleic acid and polyamide specifically bind is 1 to 100. in some embodiments from five to 50, still other embodiments from 5 to 25, and other embodiments from 5 to 20, nucleotides.

[0074] The term "cell" refers to a single cell. The term "cells" refers to a population of cells. The population may be a pure population comprising one type of cells. Likewise, the population may comprise more than one cell type. In the present disclosure, there is no limit to the number of cell types that a population of cells can comprise. A cell as used herein includes, without limitation, plant callus cells, cells with and without cell walls, prokaryotic cells and eukaryotic cells.

[0075] The term "synchronize" or "synchronized", when referring to a sample of cells, or "synchronized cells" or "synchronized cell population", refers to a plurality of cells that have been treated to cause the population of cells remained in the same phase of the cell cycle. It is not necessary for all cells in the sample to be synchronized. A small percentage of cells can be synchronized with the majority of cells in the sample. A preferable range of cells that are synchronized is between 10-100%. A more preferable range is between 30-100%. Furthermore, it is not necessary that the cells be from a pure population of a single cell type. More than one cell type may be contained in the sample. In this regard, only one of the cell types may be synchronized or may be in a different phase of the cell cycle compared to another cell type in the sample.

[0076] The term “synchronized cell”, when used in reference to a single cell, means that the cell has been manipulated so that it is in a cell cycle phase that is different from the cell cycle phase of the cell before the manipulation. Alternatively, a “synchronized cell” refers to a cell that has been manipulated to alter (i.e., increase or decrease) the length of the cell cycle phase the cell was in prior to the manipulation compared to a control cell (e.g. , a cell in the absence of manipulation).

[0077] The term “cell cycle” refers to the morphological and physiological progression of changes that the cell goes through when dividing (that is, proliferating). The cell cycle is generally recognized as being composed of phases called "interphase", "prophase", "metaphase", "anaphase" and "telophase". Additionally, the parts of the cell cycle can be called "M (mitosis)", "S (synthesis)", "G0", "G1 (interval 1)" and "G2 (interval 2)". Furthermore, the cell cycle includes periods of progression that are intermediate to the phases named above.

[0078] The term "cell cycle inhibition" refers to the cessation of cell cycle progression in a cell or population of cells. Inhibition of the cell cycle is generally induced by exposing cells to an agent (chemical, proteinaceous or other) that interferes with aspects of the cell's physiology to prevent the continuation of the cell cycle).

[0079] “Proliferation” or “cell growth” refers to the ability of a parent cell to divide into two daughter cells repeatedly, thus resulting in a total increase of cells in the population. The cell population can be in an organism or in a cultivation apparatus.

[0080] The term “capable of modifying DNA” or “means of modifying DNA” refers to procedures, as well as endogenous or exogenous agents or reagents that have the ability to induce, or can contribute to the induction of, changes in nucleotide sequence of a targeted segment of DNA. Such changes can be made by deleting, adding or replacing one or more bases in the targeted DNA segment. It is not necessary that changes in the DNA sequence confer functional changes to any gene encoded by the DNA. targeted sequence. Furthermore, it is not necessary for the DNA changes to be made to any specific part or percentage of the cells.

[0081] The term "nucleotide sequence of interest" refers to any sequence of nucleotides, the manipulation of which may be considered desired for any reason by someone skilled in the art. Such nucleotide sequences include, but are not limited to, sequences coding for structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.) and non-regulatory sequences. coding that do not encode a protein or mRNA product (e.g., promoter sequence, enhancer sequence, polyadenylation, termination sequence, regulatory RNAs such as miRNA, etc.)

[0082] "Amino acid sequence", "polypeptide sequence", "peptide sequence" and "peptide" are used interchangeably here to refer to an amino acid sequence.

[0083] "Target sequence", as used herein, refers to a double-stranded nucleic acid that comprises a sequence, preferably greater than 8 nucleotides in length, but less than 201 nucleotides in length. In some embodiments, the target sequence is preferably 8 to 30 bases. The target sequence, in general, is defined by the nucleotide sequence of one of the strands of the double helix nucleic acid.

[0084] As used herein, "purine-rich sequence" or "polypurine sequence", when used in reference to a sequence of nucleotides on one of the strands of a double helix nucleic acid sequence are defined as a contiguous sequence of nucleotides wherein more than 50% of the nucleotides of the target sequence contain a purine base. However, it is preferred that the purine-rich target sequence contains greater than 60% purine nucleotides, in some embodiments, greater than 75% purine nucleotides, in other embodiments, greater than 90% purine nucleotides. and, in yet another embodiment, 100% purine nucleotides.

[0085] As used herein, "pyrimidine-rich sequence" or "polypyrimidine sequence", when used in reference to a sequence of nucleotides on one of the strands of a double helix nucleic acid sequence are defined as a contiguous sequence of nucleotides wherein more than 50% of the nucleotides of the target sequence contain a pyrimidine base. However, it is preferred that the pyrimidine-rich target sequence contains more than 60% pyrimidine nucleotides and, in some embodiments, more than 75% pyrimidine nucleotides. In some embodiments, the sequence contains more than 90% pyrimidine nucleotides, and in other embodiments, it has 100% pyrimidine nucleotides.

[0086] A “variant” of a first nucleotide sequence is defined as a nucleotide sequence that differs from a first nucleotide sequence (e.g., by having one or more deletions, insertions, or substitutions that can be detected using assays hybridization or using DNA sequencing). This definition includes the detection of changes or modifications in the genomic sequence of the first nucleotide sequence. For example, hybridization assays can be used in order to detect (1) changes in the pattern of restriction enzyme fragments capable of hybridizing to the first nucleotide sequence when compressed into a genome (i.e., RFLP analysis), (2) the inability of a selected portion of the first nucleotide sequence to hybridize to a sample of genomic DNA containing the first nucleotide sequence (e.g., using allele-specific oligonucleotide probes), (3) inappropriate or unexpected hybridization, such as hybridization in a location other than the normal chromosomal location of the first nucleotide sequence (e.g., using fluorescence in situ hybridization (FISH) for propagation of metaphase chromosomes, etc. An example of a variant is a mutated wild-type sequence.

[0087] The terms “nucleic acid” and “unmodified nucleic acid”, as used herein, refer to any of the four known deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine and thymine). modified nucleic acid” refers to a nucleic acid whose structure is altered in relation to the structure of the unmodified nucleic acid. It would be illustrative of such modifications to replace covalent modifications of the bases, such as the alkylation of the amino and ring nitrogens. the saturation of double bonds.

[0088] As used herein, the terms "mutation" and "modification", as well as grammatical equivalents thereof, when used in reference to a nucleic acid sequence are used interchangeably to refer to a deletion, insertion, substitution, break tape and/or introduction of an adduct. A "deletion" is defined as a change in a nucleic acid sequence in which one or more nucleotides is missing. An “insertion” or “addition” is the change in a nucleic acid sequence that resulted in the addition of one or more nucleotides. A “substitution” results from the replacement of one or more nucleotides with a molecule that is a different molecule from the replaced molecule or more nucleotides. For example, a nucleic acid may be replaced by a different nucleic acid, as exemplified by the replacement of a thiamine by a cytosine, adenine, guanine or uridine. Pyrimidine to pyrimidine (e.g., nucleotide substitutions from C to T or T to C) or purine to purine (e.g., nucleotide substitutions from G to A or A to G) are called transitions, where 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 called transversions. Alternatively, a nucleic acid may be replaced by a modified nucleic acid, as exemplified by replacing a thiamine with a thiamine glycol. Mutations can result in mismatch. The term "mismatch" refers to a noncovalent interaction between two nucleic acids, each nucleic acid residing in a different polynucleic acid sequence, which does not follow the rules of base pairing. For example, for the partially complementary sequences 5-AGT-3' and 5-AAT-3', a G-A mismatch (a transition) is present. The terms “introduction of an adduct” or “adduct formation” refer to a covalent or noncovalent bond of a molecule to one or more nucleotides in a DNA sequence such that the bond results in a reduction (preferably of 10 % to 100%, more preferably from 50% to 100% and preferably, above all from 75% to 100%) at the level of DNA replication and/or transcription.

[0089] The term "DNA cutter" refers to a unit that performs a strand break. Non-limited examples include meganucleases, TALEs/TALENS, antibiotics, zinc fingers, and CRISPRS or CRISPRS/cas systems.

[0090] The term "strand break", when made in reference to a double-stranded nucleic acid sequence, includes a single-strand break and/or a double-strand break. A single-strand break (a nick) refers to an interruption in one of the two strands of the double-stranded nucleic acid sequence. This is in contrast to a double-strand break which refers to an interruption in both strands of the double-stranded nucleic acid sequence, which can result in blunt or staggered ends. Strand breaks can be introduced into the sequence of a double-stranded nucleic acid, either directly (e.g., by radiation or by treatment with certain ionizing chemicals) or indirectly (e.g., by enzymatic incision into a nucleic acid base). ).

[0091] The terms "mutant cell" and "modified cell" refer to a cell that contains at least one modification in the cell's genomic sequence.

[0092] The term “portion”, when used in reference to a nucleotide sequence, refers to fragments of that nucleotide sequence. Fragments can range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleotide residue. nucleic acid.

[0093] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to produce oligonucleotides in such a way that the 5' phosphate of the pentose ring of the mononucleotide is attached to oxygen 3 ' from its neighbor in one direction through a phosphodiester bond. Thus, one end of an oligonucleotide is designated the "5' end" if its 5' phosphate is not attached to the 3' oxygen of the pentose ring of the mononucleotide. One end of an oligonucleotide is designated the "3' end" if its 3' oxygen is not attached to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, may also be designated as having 5' and 3' ends. In a linear or circular DNA molecule, discrete elements are designated as being "upstream" or "downstream" or 3' elements. This terminology reflects that transcription continues in a 5' to 3' direction along the DNA strand. The enhancer and promoter elements that direct transcription of a linked gene are generally situated 5' or upstream of the coding region. However, potentiating elements can exert their effect even when located 3' of the promoter element and in the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0094] The term “recombinant DNA molecule”, as used herein, refers to a DNA molecule that is comprised of segments of DNA joined together using molecular biological techniques.

[0095] The term “recombinant protein” or “recombinant polypeptide”, as used herein, refers to a protein molecule that is expressed using a recombinant DNA molecule.

[0096] As used herein, the terms “vector” and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer segment(s) of DNA from one cell to another.

[0097] The terms "in operable combination", "in operable order" and "operably linked", as used herein, refer to the linking of nucleic acid sequences so that a nucleic acid molecule is capable of directing transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The terms also refer to the linking of amino acid sequences together so that a functional protein is produced.

[0098] The term "transfection", as used here, refers to the introduction of foreign DNA into cells. Transfection can be achieved by a variety of means known in the art, including DNA-calcium phosphate coprecipitation, dextran-DEAE mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofectin, protoplast fusion, retroviral infection , biolistics (i.e. particle bombardment) and the like.

[0099] As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by base pairing rules. For example, the sequence “5-CAGT-3"' is complementary to the sequence “5‘-ACTG-3‘.” Complementarity can be "partial" or "total". "Partial" complementarity is where one or more nucleic acid bases are not matched according to the rules of base pairing. "Full" or "complete" complementarity between nucleic acids is. where every nucleic acid base is matched with another base under base pairing rules. The degree of complementarity between the nucleic acid strands can have significant effects on the efficiency and strength of hybridization between the nucleic acid strands. of particular importance in amplification reactions, as well as detection methods that rely on binding between nucleic acids. For convenience, the terms "polynucleotides" and "oligonucleotides" include molecules that include nucleosides.

[0100] The terms “homology” and “homologous” as used herein in reference to nucleotide sequences refer to a level of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). When used in reference to a double-stranded nucleic acid sequence, such as cDNA or genomic clone, the term "substantially homologous" refers to any nucleic acid sequence (e.g., probe) that can hybridize to one or both of the two nucleic acid sequences. strands of double-stranded nucleic acid sequence under low stringency conditions as described above. A nucleotide sequence that is partially complementary, that is, "substantially homologous," to a nucleic acid sequence is one that at least partially inhibits one. fully complementary sequence from hybridization of a target nucleic acid sequence. Inhibition of hybridization of the fully complementary sequence to the target sequence can be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under low conditions. rigor. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., hybridization) of a completely homologous sequence to a target sequence under low stringency conditions. This is not to say that the low stringency conditions are such that nonspecific binding is not permitted; Low stringency conditions require that the binding of two sequences to each other be a specific (i.e., selective) interaction. The absence of nonspecific binding can be tested by using a second target sequence without even a partial level of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding, the probe will not hybridize to the second noncomplementary target.

[0101] Low stringency conditions comprise conditions equivalent to binding or hybridization at 68 °C in a solution consisting of 5xSSPE (43.8 g/| NaCl, 6.9 g/l Na^POrH∑O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardt's reagent (50x Denhardt contents per 500 ml: 5 g of Ficoll (type 400, Pharmacia), 5 g of BSA (fraction V; Sigma)) and 100 μg/ ml of denatured salmon semen 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.

[0102] Furthermore, conditions that promote hybridization under conditions of high intolerance (e.g., increasing the hybridization temperature and/or washing steps, the use of formamide in the hybridization solution, etc.) are well known in the art . High intolerance conditions, when used in reference to nucleic acid hybridization, comprise conditions equivalent to ligation or hybridization at 68°C in a solution consisting of 5xSSPE, 1% SDS, 5x Denhardt's reagent, and 100 μg/ml of Denatured salmon semen DNA followed by washing in a solution comprising 0.1*SSPE and 0.1% SDS at 68°C when a probe of about 100 to about 1000 nucleotides in length is employed.

[0103] It is well known in the art that numerous equivalent conditions can be employed to understand low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the round and the nature of the target (DNA, RNA, base composition, present in solution 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 solution can be varied to generate low stringency hybridization conditions different from, but equivalent to, the conditions listed above.

[0104] The term "equivalent", when used in reference to the hybridization condition, as it relates to a hybridization condition of interest, means that the hybridization condition and the hybridization condition of interest result in the hybridization of the nucleic acid sequences that have the same percentage range (%) of homology. For example, if a hybridization condition of interest results in the hybridization of a first nucleic acid sequence with other nucleic acid sequences that have 50% to 70% homology to the first nucleic acid sequence, then another hybridization condition is equivalent to the hybridization condition of interest if that other hybridization condition also results in hybridization of the first nucleic acid sequence with other nucleic acid sequences having 50% to 70% homology to the first nucleic acid sequence.

[0105] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process by which a nucleic acid strand joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of association between the nucleic acids) are impacted by these factors, as well as the degree of complementarity between the nucleic acids, the intolerance of the conditions implicated, the Tm of the hybrid formed, and the G:C ratio in nucleic acids.

[0106] As used herein, the term "hybridization complex" refers to a complex formed between two nucleic acid sequences due to the formation of hydrogen bonds between the complementary bases G and C and between the complementary bases A and T ; these hydrogen bonds can be further stabilized by base stacking interactions. The two complementary nucleic acids sequence the hydrogen bond in an antiparallel configuration. A hybridization complex can be formed in solution (e.g., Cot or Rot analysis) or between a nucleic acid sequence present in the solution and another nucleic acid sequence immobilized on a solid support (e.g., a nylon membrane or a filter nitrocellulose as used in Southern and Northern blotting, dot blotting, or a glass slide as used in in situ hybridization, including FISH (fluorescence in situ hybridization)).

[0107] As used herein, the term "Tm" is used in reference to "melting temperature". Melting temperature is the temperature at which a population of double-stranded nucleic acid molecules is split into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the value of Tm can be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution in 1 M NaCl (see, for example, Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization, 1985). Other references include more sophisticated computations that take into account structural as well as sequential features for calculating Tm.

[0108] As used herein, the term "stringency" is used in reference to the conditions of temperature, ionic strength and the presence of other compounds, such as organic solvents, under which nucleic acid hybridizations are conducted. "Stiffness" typically occurs in a range of about Tm-5°C (5°C below probe melting temperature) to about 20°C to 25°C below Tm. As will be understood by those skilled in the art, stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.

[0109] The terms "specific binding", "binding specificity" and grammatical equivalents thereof, when used in reference to the binding of a first nucleotide sequence to a second nucleotide sequence, refer to the preferential interaction between the first nucleotide sequence nucleotides and the second nucleotide sequence compared to the interaction between the second nucleotide sequence and the third nucleotide sequence. Specific binding is a relative term that does not require an absolute specificity of binding: put another way, the term "specific binding" does not require that the second nucleotide sequence interact with the first nucleotide sequence in the absence of an interaction between the second nucleotide sequence and the third nucleotide sequence. It is sufficient that the level of interaction between the first nucleotide sequence and the second nucleotide sequence is greater than the level of interaction between the second nucleotide sequence and the third nucleotide sequence. "Specific binding" of a first nucleotide sequence to a second nucleotide sequence also means that the interaction between the first nucleotide sequence and the second nucleotide sequence depends on the presence of a specific structure on or in the first nucleotide sequence; put another way, the second nucleotide sequence is recognizing and binding to a specific structure on or in the first nucleotide sequence rather than to nucleic acids or nucleotide sequences in general. For example, if a second nucleotide sequence is specific to the "A" structure that is on or in the first nucleotide sequence, the presence of a third nucleotide sequence containing the A structure will reduce the amount of the second nucleotide sequence that is bound to the first nucleotide sequence.

[0110] As used herein, the term "amplifiable nucleic acid" is used in reference to nucleic acids that can be amplified by any amplification method. It is contemplated that the "amplifiable nucleic acid" will generally comprise the "sample template".

[0111] The terms "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotide sequence that is linked to a nucleic acid sequence that is unlinked in nature or to which it is linked in a different place in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but was obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Examples of heterologous DNA include reporter genes, translational and transcriptional regulatory sequences, selectable marker proteins (e.g., proteins that confer drug resistance), etc.

[0112] “Amplification” is defined as the production of additional copies of a nucleic acid sequence and is generally performed using polymerase chain reaction technologies well known in the art (Dieffenbach C W and G S Dveksler (1995) PCR Primer, a LaboratoryManual , Cold Spring Harbor Press, Plainview, N.Y.) As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of U.S. K. B. Mullis Patent Nos. 4,683,195 and 4,683,202, incorporated herein 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 amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers relative to each other, and thus this length is a controllable parameter. Because of the repetitive aspect of the process, the method is referred to as “polymerase chain reaction” (“PCR”). Since the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified".

[0113] With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by detection of the avidin- enzyme; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, in themselves, efficient templates for subsequent PCR amplifications.

[0114] One such preferable method, particularly for commercial applications, is based on the widely used TaqMan® real-time PCR technology and combines allele-specific PCR with a blocking reagent (ASB-PCR) to suppress amplification of the wild-type allele . ASB-PCR can be used to detect germline or somatic mutations in DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor samples. A set of reagent design rules are developed to enable the sensitive and selective detection of single point substitutions, insertions, or selections against a wild-type allele background in a thousand-fold excess or more. (Morlan J, Baker J, Sinicropi D Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. PLoS ONE 4(2): e4584, 2009)

[0115] The terms "reverse transcription polymerase chain reaction" and "RT-PCR" refer to a method of reverse transcription of an RNA sequence to generate a mixture of cDNA sequences, followed by increasing the concentration of a desired segment of the transcribed cDNA sequences in the mixture, without cloning or purification. Typically, RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of DNA transcribed using two primers.

[0116] As used herein, the term "primer" refers to an oligonucleotide, naturally occurring, as in a purified restriction digest, or synthetically produced, that is capable of acting as a synthesis initiation point when placed under conditions in which the synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a temperature and pH appropriate). In some embodiments, the primer is preferably single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. If it is double stranded, the primer is first treated to separate its strands before they are used to prepare the extension products. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, origin of the primer, and method use.

[0117] As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by amplification of PCR, which is capable of hybridizing another oligonucleotide of interest. The probe can be single-stranded or double-stranded. Probes are useful in detecting, identifying and isolating specific genetic sequences. It is contemplated that every probe used in the present disclosure will be labeled as any "reporter molecule" so that it is detectable in any detection system, including, but not limited to, luminescent, radioactive, fluorescent, and enzyme systems (e.g., ELISA as well as enzyme-based histochemical assays). The present disclosure is not intended to be limited to any specific label or detection system.

[0118] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes that cut or incise double- or single-stranded DNA at or near a specific nucleotide sequence, e.g., a endonuclease domain of a type IIS restriction endonuclease (e.g., Fokl) may be used, as taught by Kim et al., 1996, Proc. Nat'l. Academic. Sci. USA, 6:1 156-60).

[0119] 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, that is, the nucleic acid sequence encoding a product of the gene. The coding region may be present in a cDNA, genomic DNA, or RNA format. When present in a DNA format, the oligonucleotide can be single-stranded (i.e., the sense strand) or double-stranded. Additionally, "an oligonucleotide with an oligonucleotide sequence encoding a gene" may include appropriate control elements, such as enhancers, promoters, splice junctions, polyadenylation signals, etc., if necessary to allow for proper initiation of transcription and/or processing. In addition, the coding region of the present disclosure may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, etc.

[0120] Transcriptional control signals in eukaryotes comprise "enhancer" elements. Enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription (Maniatis, T. et al., Science 236:1237, 1987). Enhancer elements have been isolated from a variety of eukaryotic sources, including genes from viruses and plant, yeast, insect, and mammalian cells. The selection of a specific enhancer depends on what type of cell is to be used to express the protein of interest.

[0121] The presence of "splicing signals" in an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and an acceptor site (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press , New York, pp. 16.7-16.8, 1989). A commonly used splice acceptor and donor site is the SV40 16S RNA splice junction.

[0122] The efficient expression of recombinant DNA sequences in eukaryotic cells requires the expression of signals that direct polyadenylation and the efficient termination of the resulting transcription. Transcription termination signals are usually 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 that directs the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts without the poly A tail are unstable and are rapidly degraded. The poly A signal used in an expression vector can be “heterologous” or “endogenous”. An endogenous polyA signal is one found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that is isolated from one gene and placed 3' of another gene.

[0123] The terms "promoter", "promoter element" or "promoter sequence", as used herein, refer to a DNA sequence that, when placed at the 5' end of (i.e., precedes) an oligonucleotide sequence , is capable of controlling the transcription of the oligonucleotide sequence to mRNA. A promoter is typically positioned 5' (i.e., upstream) of an oligonucleotide sequence whose transcription to mRNA it controls and provides a site for specific binding by RNA polymerase and initiation of transcription.

[0124] The term "promoter activity", when used in reference to a nucleic acid sequence, refers to the ability of the nucleic acid sequence to initiate transcription of an oligonucleotide sequence into mRNA.

[0125] The term “tissue-specific”, as applied to a promoter, refers to a promoter that is capable of directing selective expression of an oligonucleotide sequence to a specific type of tissue in the relative absence of expression of the same oligonucleotide in a different type of tissue. The tissue specificity of a promoter can be assessed by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of an animal or plant so that the reporter construct be integrated into each tissue of the resulting transgenic animal and detection of reporter gene expression (e.g., detection of mRNA, protein, or activity of a protein encoded by a reporter gene) in different tissues of the transgenic animal or plant. Selectivity does not need to be absolute. The detection of a higher level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific to the tissues in which higher levels of expression are detected.

[0126] The term “cell-specific”, as applied to a promoter, refers to a promoter that is capable of directing selective expression of an oligonucleotide sequence to a specific cell type in the relative absence of expression of the same sequence of oligonucleotides in a different type of cell in the same tissue. The term "cell type specific", when applied to a promoter, also denotes a promoter capable of promoting selective expression of an oligonucleotide in a region in a single tissue. Again, selectivity need not be absolute. The cell type specificity of a promoter can be assessed using methods well known in the art, for example, immunohistochemical staining, as described herein. Briefly, tissue sections are fixed in paraffin and the paraffin sections are reacted with a primary antibody that is specific for the polypeptide product encoded by the oligonucleotide sequence whose expression is controlled by the promoter. As an alternative to paraffin sections, samples can be cryosectioned. For example, sections can be frozen before and during sectioning, thus avoiding potential interference by residual paraffin. A labeled secondary antibody (e.g., peroxidase conjugate) that is specific to the primary antibody is allowed to bind to the sectioned tissue and the specific binding detected (e.g., with avidin/biotin) by microscope.

[0127] The terms “selective expression”, “selectively express” and grammatical equivalents thereof refer to a comparison of relative levels of expression in two or more regions of interest. For example, “selective expression,” when used in connection with tissues, refers to a substantially greater level of expression of a gene of interest in a specific tissue or to a substantially greater quantity of cells expressing the gene in that tissue compared to , respectively, with the level of expression and the number of cells that express the same gene in another tissue (i.e., selectivity does not need to be absolute). Selective expression does not require, although it may include, expression of a gene of interest in a specific tissue and a total absence of expression of the same gene in another tissue. Similarly, “selective expression,” as used herein in reference to cell types, refers to a substantially higher level of expression of, or a substantially higher number of cells that express, a gene of interest in a specific cell type in comparison, respectively, to gene expression levels and the number of cells expressing the gene in another cell type.

[0128] The term “contiguous”, when used in reference to two or more nucleotide sequences, means that the nucleotide sequences are linked in tandem in the absence of intervening sequences or in the presence of intervening sequences that do not comprise one or more control elements.

[0129] As used herein, the terms "encoding nucleic acid molecule", "encoding nucleotide", "encoding DNA sequence" and "encoding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribocleic acid ribbon. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes the amino acid sequence.

[0130] The term "isolated", when used in relation to a nucleic acid, such as an "isolated oligonucleotide", refers to a nucleic acid sequence that is separate from at least one contaminating nucleic acid with which it is normally associated in its natural source. Isolated nucleic acid is nucleic acid present in a format or configuration different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state in which they exist in nature. For example, a particular DNA sequence (e.g., a gene) is found on the host cell's chromosome in close proximity to neighboring genes; RNA sequences, such as the specific mRNA sequence that codes for a specific protein, are found in the cell as a mixture with numerous other mRNAs that code for multiple proteins. However, isolated nucleic acid encoding a polypeptide of interest includes, by way of example, such nucleic acid in cells that normally express the polypeptide of interest, wherein the nucleic acid is in a chromosomal or extrachromosomal location different from that in natural cells or along with a sequence of nucleic acids different from that found in nature. The isolated nucleic acid or oligonucleotide may be present in a single-stranded or double-stranded format. The isolated nucleic acid can be readily identified (if desired) by a variety of techniques (e.g., hybridization, dot blotting, etc.). When a nucleic acid or an oligonucleotide is to be used to express a protein, the oligonucleotide will contain at least the coding or sense strand (i.e., the oligonucleotide may be single-stranded). Alternatively, it may contain both sense and antisense strands (i.e., the oligonucleotide may be double-stranded).

[0131] As used herein, the term "purified" or "to purify" refers to the removal of one or more (unwanted) components from a sample. For example, where recombinant polypeptides are expressed in host bacterial cells, the polypeptides are purified by removing the host cell proteins, thereby increasing the percentage of recombinant polypeptides in the sample.

[0132] As used herein, the term "substantially purified" refers to molecules, amino acid sequences or nucleic acids, that are removed from their natural environment, isolated or separated and are at least 60% free, in some embodiments, 75% free and, in other modalities, 90% free of other components with which they are naturally associated. An "isolated polynucleotide" is thus a substantially purified polynucleotide.

[0133] As used herein, the term "coding region", when used in reference to a structural gene, refers to the nucleotide sequences that encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is linked, in eukaryotes, on the 5' side, usually by the nucleotide triplet "ATG", which encodes the initiator methionine, and on the 3' side, by one of the three triplets that specify the termination codons (i.e. TAA, TAG, TGA).

[0134] 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 and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are spliced together by the cell's biochemical machinery to provide a mature mRNA. The antisense strand is the nucleic acid complement, and the coding sequence can be deduced from this.

[0135] By "non-coding sequence" is meant a nucleic acid sequence or its complement, or a part thereof, that is not transcribed into amino acid in vivo, or where the tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.

[0136] As used herein, the term "structural gene" or "structural nucleotide sequence" refers to a DNA sequence that codes for RNA or a protein that does not control the expression of other genes. In contrast, a "regulatory gene" or a "regulatory sequence" is a structural gene that encodes products (e.g., transcription factors) that control the expression of other genes.

[0137] As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription from an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

[0138] As used herein, the term "peptide transcription factor binding site" or "transcription factor binding site" refers to a nucleotide sequence that binds protein transcription factors and thus controls some aspect of the expression of nucleic acid sequences. For example, the Sp-1 and AP1 (activator protein 1) binding sites are examples of peptide transcription factor binding sites.

[0139] As used herein, the term "gene" means the deoxyribonucleotide sequences that comprise the coding region of a structural gene. A “gene” may also include untranslated sequences located adjacent to the coding region at the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. Sequences that are situated 5' of the coding region and are present in the mRNA are referred to as 5'-untranslated sequences. Sequences that are situated 3' or downstream of the coding region and that are present in the mRNA are referred to as 3' untranslated sequences. The term “gene” encompasses both the genomic and cDNA forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences called "introns", "intervening regions" or "intervening sequences". Introns are segments of a gene that are written in heterogeneous nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed ("spliced out") from the primary or nuclear transcript; Introns are thus absent from messenger RNA (mRNA) transcription. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. A gene is generally a single locus. In a normal diploid organism, a gene has two alleles. In tetraploid potatoes, however, each gene has 4 alleles. In sugarcane, which is dodecaploid, there can be 12 alleles per gene. Particular examples include flax, which has two EPSPS loci each with two alleles, and rice, which has a single homomeric plastid ACCase with two alleles.

[0140] In addition to containing introns, the genomic forms of a gene may also include sequences located at the 5' and 3' ends of the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences lie at 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences, such as promoters and enhancers, that control or influence gene transcription. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.

[0141] A “non-human animal” refers to any animal that is not a human and includes vertebrates, such as rodents, non-human primates, sheep, cattle, ruminants, lagomorphs, swine, goats, horses, canines, felines, birds , etc. Preferable non-human animals are selected from the order Rodentia. "Non-human animal" additionally refers to amphibians (e.g., Xenopus), reptiles, insects (e.g., Drosophila), and other non-mammalian animal species. .

[0142] As used herein, the term "transgenic" refers to an organism or cell that has DNA derived from another organism within which it becomes integrated into the genome of the somatic and/or germline cells of the plant or animal . A “transgene” means a DNA sequence that is partially or completely heterologous (i.e., not present in nature) to that animal or plant in which it is found or that is homologous to an endogenous sequence (i.e., a sequence that is found in the animal in nature) and is inserted into the animal or plant genome at a location different from that of the naturally occurring sequence. Transgenic plants or animals that include one or more transgenes are within the scope of this disclosure. Additionally, a “transgenic,” as used herein, refers to an organism that has one or more genes modified and/or “knocked out” (rendered nonfunctional or made to function at a reduced level, i.e., a “knockout” mutation). ") by the methods of the disclosure, by homologous recombination, TFO mutation or similar processes. For example, in some embodiments, a transgenic cell or organism includes an inserted DNA that includes a foreign promoter and/or a coding region.

[0143] A “transformed cell” is a cell or cell line that has acquired the ability to grow in cell culture for multiple generations, the ability to grow in light agar and/or the ability to not have cell culture inhibited by cell contact with cell. In this regard, transformation refers to the introduction of foreign genetic material into an organism or cell. Transformation can be carried out by any known method that allows the successful introduction of nucleic acids into cells and which results in expression of the introduced nucleic acid. “Transformation” includes, but is not limited to, methods such as transfection, microinjection, electroporation, nucleofection, and lipofection (liposome-mediated gene transfer). The transformation can be performed using any expression vector. For example, the use of baculovirus to introduce foreign nucleic acid into infected cells is contemplated. The term “transformation” further includes methods such as P-element-mediated germline transformation of all insects. Additionally, transformation refers to cells that have been naturally transformed, usually through genetic mutation.

[0144] As used herein, "exogenous" means that the gene encoding the protein is not normally expressed in the cell. Additionally, "exogenous" refers to a gene transfected into a cell to increase the normal (i.e., natural) level of expression of that gene.

[0145] The peptide and nucleotide sequence can be "endogenous" or "heterologous" (i.e., "foreign"). The term "endogenous" refers to a sequence that is found naturally in the cell into which it is introduced, as long as it does not contain modifications relative to the naturally occurring sequence. The term "heterologous" refers to a sequence that is not endogenous to the cell into which it is introduced. For example, heterologous DNA includes a nucleotide sequence that is linked to, or is engineered to be linked to, a nucleic acid sequence that is not naturally linked or that is not linked at a different location in nature. Heterologous DNA further includes a nucleotide sequence that is found naturally in the cell into which it is introduced and that contains modifications 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 heterologous DNA include reporter genes, translational and transcriptional regulatory sequences, DNA sequences encoding selectable marker proteins (e.g., proteins that confer drug resistance), etc.

[0146] Constructs

[0147] The nucleic acid molecules disclosed herein (e.g., site-specific nucleases or guidance RNA for CRISPRSs) can be used in the production of the recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the present disclosure can be used in the preparation of nucleic acid constructs, for example, in expression cassettes for expression in the plant of interest. This expression may be transient, for example, when the construct is not integrated into the host genome or maintained under the control offered by the promoter and at the position of the construct in the host genome if it becomes integrated.

[0148] Expression cassettes may include regulatory sequences operably linked to the site-specific nuclease or guidance RNA sequences disclosed herein. The cassette may additionally contain at least one additional gene to be cotransformed in the organism. Alternatively, the additional gene(s) may be provided in multiple expression cassettes.

[0149] The nucleic acid constructs can be provided with a plurality of restriction sites for insertion of the site-specific nuclease coding sequence to be under the transcriptional regulation of the regulatory regions. The nucleic acid constructs may additionally contain nucleic acid molecules that encode selectable marker genes.

[0150] Any promoter can be used in the production of nucleic acid constructs. The promoter may be native or analogous, or foreign or heterologous, to the host nucleic acid sequences of the plant, microbe or animal disclosed herein. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "foreign" or "heterologous" to the plant, microbial or animal host it is intended that the promoter will not be found in the native plant, microbe or animal into which the promoter is introduced. As used herein, a chimeric gene comprises a coding sequence operatively linked to a transcription initiation region that is heterologous to the coding sequence.

[0151] The site-directed nuclease sequences disclosed herein can be expressed using heterologous promoters.

[0152] Any promoter can be used in the preparation of constructs to control the expression of site-directed nuclease sequences, such as promoters that provide constitutive, tissue-preferable, inducible, or other promoters for expression in plants, microbes, or animals. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Patent and WO 99/43,838 No. 6,072,050; from the CaMV 35S core promoter (Odell et al. Nature 313:810-812; 1985); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); ubiquitin (Christensen et al., Plant Mol. 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); BUT (Velten et al., EMBO J. 3:2723-2730, 1984); ALS promoter (U.S. Patent No. 5,659,026) and the like. Other constituent promoters include, for example, U.S. Patent No. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0153] Tissue-preferable promoters can be used to direct site-directed nuclease expression in a specific plant tissue. Such tissue-preferable promoters include, but are not limited to, leaf-preferable promoters, root-preferable promoters, and stem-preferable promoters. Preferable promoters for tissue include Yamamoto et al., Plant J. 12(2):255-265, 1997; Kawamata et al., Plant CellPhysiol. 38(7):792-803, 1997; Hansen et al., Mol. Gen Genet. 254(3):337-343, 1997; Russell et al., Transgenic Res. 6(2):157-168, 1997; Rinehart et al., Plant Physiol. 1 12(3):1331-1341, 1996; Van Camp et al., Plant Physiol. 1 12(2):525-535, 1996; Canevascini et al., Plant Physiol. 112(2): 513-524, 1996; Yamamoto et al., Plant Cell Physiol. 35(5):773-778, 1994; Lam, Results Probl. Cell Difference. 20:181-196, 1994; Orozco et al. Plant Mol Biol. 23(6):1129-1138, 1993; Matsuoka et al., Proc Nat’l. Academic. Sci. USA 90 (20): 9586 - 9590, 1993; and Guevara-Garcia et al., Plant J. 4 (3): 495-505, 1993.

[0154] Nucleic acid constructs may further include transcription termination regions. Where transcription termination regions are used, any termination region can be used in preparing the nucleic acid constructs. For example, the termination 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 of the A. tumefaciens Ti-plasmid, such as octopine synthase and nopaline synthase termination regions. See also Guerineau et al., Mol. Gen Genet. 262:141-144, 1991; Proudfoot, Cell 64:671-674, 1991; Sanfacon et al., Genes Dev. 5:141-149, 1991; Mogen et al., Plant Cell 2:1261-1272, 1990; Munroe et al., Gene 91:151-158, 1990; Ballas et al., Nucleic Acids Res. 17:7891-7903, 1989; and Joshi et al., Nucleic Acid Res. 15:9627-9639, 1987.

[0155] Together with any of these aspects, the modalities, methods and/or compositions disclosed herein, nucleic acids can be optimized for increased expression in the transformed plant. That is, nucleic acids encoding site-directed nuclease proteins can be synthesized using plant-preferable codons for increased expression. See, for example, Campbell and Gowri, (Plant Physiol. 92:1-11, 1990) for a discussion of host-preferable codon usage. Methods are available in the art to synthesize preferable genes for the plant. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al., Nucleic Acids Res. 17:477-498, 1989. See also, for example, Lanza et al., BMC Systems Biology 8 :33-43, 2014; Burgess-Brown et al., Protein Expr. Purif. 59:94-102, 2008; Gustafsson et al., Trends Biotechnol 22:346-353, 2004.

[0156] Furthermore, other sequence 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. They include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and other well-characterized sequences of these that may be deleterious to gene expression. The G-C content of the sequence can also be adjusted to average levels for a target cell host, as calculated by reference to known genes expressed in the host cell. Furthermore, the sequence can be modified to avoid hairpin secondary mRNA structures.

[0157] Other nucleic acid sequences can also be used in preparing the constructs of the present disclosure, for example, to enhance the expression of the site-directed nuclease coding sequence. Such nucleic acid sequences include maize Adhl introns, intron gene (Callis et al., Genes and Development 1:1183-1200, 1987) and tobacco mosaic virus (TMV) leader sequences (W-sequence), corn chlorotic stunt virus and alfalfa mosaic virus (Gallie et al., Nucleic Acid Res. 15:8693-8711, 1987; and Skuzeski et al., Plant Mol. Biol. 15:65-79, 1990). Maize first shrunken site-1 has been shown to increase gene expression in chimeric gene constructs. US Patent Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs and Gallie et al. (Plant Physiol. 106:929-939, 1994) also showed that introns are useful for regulating gene expression on a tissue-specific basis. To further enhance or optimize targeted nuclease gene expression, the plant expression vectors disclosed herein may also contain DNA sequences containing matrix docking regions (MARs). Transformed plant cells with such modified expression systems may then exhibit overexpression or constitutive expression of a nucleotide sequence of the disclosure.

[0158] The expression constructs disclosed herein may also include nucleic acid sequences capable of directing the expression of the site-directed nuclease sequence to the chloroplast or other organelles and structures in prokaryotes and eukaryotes. Such nucleic acid sequences include chloroplast targeting sequences that encode a chloroplast transit peptide to direct the gene product of interest to the chloroplasts of the plant cell. Such transit peptides are known in the art. With respect to chloroplast targeting sequences, "operationally linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast targeting sequence) is linked to the disclosed site-directed nuclease nucleic acid molecules. here, so that two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993; and Shah et al., Science 233:478-481, 1986.

[0159] Chloroplast target sequences are known in the art and include the small chloroplast subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al., Plant Mol. Biol. 30:769 -780, 1996; Schnell et al., J. Biol. 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(11):6081-6087, 1995); plastocyanin (Lawrence et al., J. Biol. Chem. 272(33):20357-20363, 1997); chorismate synthase (Schmidt et al., J. Biol. Chem. 268(36):27447-27457, 1993); and light-harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al., J. Biol. Chem. 263:14996-14999, 1988). See also Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993; and Shah et al., Science 233:478-481, 1986.

[0160] In conjunction with any of the aspects, modalities, methods and/or compositions disclosed herein, nucleic acid constructs can be prepared to direct expression of the mutant site-directed nuclease coding sequence from the chloroplast of the plant cell . Methods for transforming chloroplasts are known in the art. See, for example, Svab et al., Proc. Nat'l. Academic. Sci. USA 87:8526-8530, 1990; Svab and Maliga, Proc. Nat'l. Academic. Sci. USA 90:913-917, 1993; Svab and Maliga, EMBO J. 12:601-606, 1993. The method is based on biolistic delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Furthermore, plastid transformation can be accomplished by transactivation of a plastid-borne silent transgene by tissue-preferable expression of a plastid-directed, nuclear-encoded RNA polymerase. Such a system was reported in McBride et al. Proc. Nat'l. Academic. Sci USA 91:7301-7305, 1994.

[0161] Nucleic acids of interest to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for differences in codon usage between this organelle and the plant nucleus. In this way, the nucleic acids of interest can be synthesized using chloroplast-preferable codons. See for example, U.S. Patent No. 5,380,831 herein incorporated by reference.

[0162] Nucleic acid constructs can be used to transform plant cells and regenerate transgenic plants that comprise site-directed nuclease coding sequences. Numerous vectors and plant transformation methods are available. See, for example, U.S. Patent No. 6,753,458, An, G. et al., Plant Physiol., 81:301-305, 1986; Fry, J. et al., PlantCell Rep. 6:321-325, 1987; Block, M., Theor. Appl Genet. 76:767-774, 1988; Hinchee et al., Stadler. 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/Technol. 10:309-314, 1992; Dhir et al., Plant Physiol. 99:81-88, 1992; Casas et al., Proc. Nat'l. Acad Sci. USA 90:11212-11216, 1993; Christou, P., In Vitro Cell. Dev. Biol.-Plant 29P:1 19-124, 1993; Davies, et al., Plant Cell Rep. 12:180-183, 1993; Dong, J. A. and Mc Hughen, A., Plant Sci. 91:139-148, 1993; Franklin, 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, et al., 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. 5:583592, 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 et al., Plant Cell Rep. 13:582-586, 1994; Hartman et al., Bio-Technology 12:919923, 1994; Ritala et al., Plant. Mol. Biol. 24:317-325, 1994; and Wan, Y. C. and Lemaux, P. G., Plant Physiol. 104:3748, 1994. The constructs can also be transformed into plant cells using homologous recombination.

[0163] The term “wild type”, when used in reference to a peptide sequence and a nucleotide sequence, refers to a peptide sequence and a nucleotide sequence (locus/gene/allele), respectively, that have characteristics of that sequence peptide and nucleotide sequence when isolated from a naturally occurring source. A wild-type peptide and nucleotide sequence is one that is observed most frequently in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the peptide sequence and nucleotide sequence, respectively. "Wild-type" may also refer to the sequence at a specific nucleotide position, or positions, or to the sequence at a specific codon position, or positions, or to the sequence at a specific amino acid position, or positions.

[0164] "Consensus sequence" is defined as a sequence of amino acids or nucleotides that contains identical amino acids or nucleotides or functionally equivalent nucleotides or amino acids for at least 25% of the sequence. Identical or functionally equivalent nucleotides or amino acids need not be contiguous.

[0165] The term "Brassica", as used herein, refers to plants of the genus Brassica. Exemplary species of the Brassica species include, but are not limited to, B. carinata, B. elongate, B. fruticulosa, B. juncea, B. napus, B. narinosa, B. nigra, B. oleracea, B. perviridis, B. rapa (syn B. campestris), B. rupestris, B. septiceps and B. tournefortii.

[0166] A nucleobase is a base, which in certain preferred embodiments is a purine, pyrimidine or a derivative or analogue thereof. Nucleosides are nucleobases that contain a pentosefuranosyl unit, for example, an optionally substituted riboside or 2'-deoxyriboside. Nucleosides can be linked by one of several linking moieties, which may or may not contain phosphorus. Nucleosides that are linked by unsubstituted phosphodiester bonds are called nucleotides. The term "nucleobases" as used herein includes peptide nucleobases, the nucleic acid subunits of peptides and morpholine nucleoses, as well as nucleosides and nucleotides.

[0167] An oligonucleobase is a polymer that comprises nucleobases; in some embodiments, at least a portion of which hybridizes by Watson-Crick base pairing to a DNA containing the complementary sequence. A chain of oligonucleobases can have a single 5' and 3' terminus, which are the last nucleobases of the polymer. A specific oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that can be complementary and hybridized by Watson-Crick base pairing. Ribo-type nucleobases include pentosefuranosyl-containing nucleobases in which the 2' carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases, and include all nucleobases that do not contain a pentosefuranosyl unit.

[0168] In certain embodiments, an oligonucleobase strand may include oligonucleobase chains and segments or regions of oligonucleobase 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' ends of the chain.

[0169] As used herein, the term "codon" refers to a sequence of three adjacent nucleotides (RNA or DNA) that constitute the genetic code that determines the insertion of a specific amino acid into a polypeptide chain during protein synthesis or that the signal stops protein synthesis. The term "codon" is also used to refer to the corresponding (and complementary) sequences of three nucleotides in the messenger RNA into which the original DNA is transcribed.

[0170] As used herein, the term "homology" refers to the sequence similarity between 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 100% sequence identity compared to another sequence.

[0171] "Heterozygous" refers to having different alleles at one or more genetic sites on homologous chromosomal segments. As used herein, "heterozygote" may also refer to a sample, cell, cell population, or organism in which different alleles at one or more genetic sites can be detected. Heterozygous samples can also be determined using methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows two peaks at a single location and both peaks are strictly the same size, the sample can 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 can be characterized as homozygous. In some embodiments, the smaller peak is at least about 15% the size of the larger peak. In other embodiments, the smaller peak is at least about 10% the size of the larger peak. In other embodiments, the smaller peak is at least about 5% the size of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

[0172] As used herein, "homozygous" refers to having identical alleles at one or more genetic sites on homologous chromosomal segments. "Homozygous" may also refer to a sample, cell, cell population, or organism in which the same alleles at one or more genetic sites can be detected. Homozygous samples can also be determined using methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a specific location, the sample may be called "homozygous" for that location.

[0173] The term "hemizygous" refers to a gene segment or gene being present only once in the genotype of a cell or an organism because the second allele is deleted, or not present in the homologous chromosome segment. As used herein "hemizygous" may also refer to a sample, a cell, a population of cells or an organism in which an allele of one or more genetic loci can be detected only once in the genotype.

[0174] The term "zygosity status", as used herein, refers to a sample, a population of cells, or an organism as appearing heterozygous, homozygous, or hemizygous as determined by assay methods known in the art and described herein. . The term "zygosity status of a nucleic acid" means determining whether the source nucleic acid appears heterozygous, homozygous, or hemizygous. "Zygosity status" can refer to differences in single nucleotide position in a sequence. In some methods, the zygosity status of a sample with respect to a single mutation can be categorized as wild-type homozygous, 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 allele).

[0175] As used herein, the term "RTDS" refers to The Rapid Trait Development System™ (RTDS) developed by Cibus. RTDS is a site-specific genetic modification system that is effective in making precise changes to a gene sequence without incorporating foreign genes or control sequences.

[0176] The term "about" as used herein means in quantitative terms plus or minus 10%. For example, "about 3%" would cover 2.7-3.3% and "about 10%" would cover 9-11%. Furthermore, where "about" is used here in conjunction with a quantitative term it is understood that, in addition to the value of plus or minus 10%, the exact value of the quantitative term is also contemplated and described. For example, the term "about 3%" expressly contemplates, describes and includes exactly 3%.

[0177] RTDS and Repair Oligonucleotides (GRONs)

[0178] This disclosure generally refers to new methods for improving the efficiency of targeting modifications to specific locations in genomic sequences or other nucleotide sequences. Furthermore, this disclosure refers to target DNA that has been modified, mutated, or marked by the approaches disclosed herein. The disclosure also relates to cells, tissue and organisms that have been modified by the methods of the invention. The present disclosure is based on the development of compositions and methods relating, in part, to a successful conversion system, the Rapid Trait Development System (RTDS™, Cibus US LLC).

[0179] RTDS is based on altering a targeted gene using the cell's own gene repair system to specifically modify the genetic sequence in situ and not insert foreign DNA and gene expression control sequences. This procedure makes a precise change in the genetic sequence, while the rest of the genome is left unchanged. In contrast to conventional transgenic GMOs, there is no integration of foreign genetic material, nor is any foreign genetic material left in the plant. Genetic sequence changes introduced by RTDS are not inserted randomly. Since the affected genes remain in their native location, no adverse, uncontrolled, or random pattern of expression occurs.

[0180] The RTDS that effects this change is a chemically synthesized oligonucleotide (GRON), as described herein, which can be composed of DNA and modified RNA bases, as well as other chemical moieties, and is designed to hybridize at the gene site. desired to create misfit base pair(s). This mismatched base pair acts as a signal to attract the cell's own natural gene repair system to that location and correct (replace, insert, or delete) the designated nucleotide(s) in the gene. Once the correction process is complete, the RTDS molecule is degraded and the newly modified or repaired gene is expressed under the gene's normal endogenous control mechanisms.

[0181] The methods and compositions disclosed herein can be practiced or made with "gene repair oligonucleobases" (GRON) having the conformations and chemical substances as described in detail below. "Gene repair oligonucleobases" as contemplated herein have also been described in published scientific and patent literature using other names, which include "recombinogenic oligonucleobases"; "chimeric RNA/DNA oligonucleotides; "chimeric oligonucleotides"; "mixed duplex oligonucleotides" (MDONs); "RNA/DNA oligonucleotides" (RDOs); "gene-targeted oligonucleotides"; "genoplasts"; single-strand modified oligonucleotides "; "single-stranded oligodeoxynucleotide mutational vectors (SSOMVs)"; "duplex 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 (biolistic delivery), microfibers, polyethylene glycol (PEG)-mediated absorption, electroporation and microinjection.

[0182] In one embodiment, the gene repair oligonucleobase is a mixed duplex oligonucleotide (MDON) in which the RNA-like nucleotides of the mixed duplex oligonucleotide are made resistant to RNase by replacing the 2'-hydroxyl with a fluoro functionality, chlorine or bromine or by placing a substituent on 2'-O. Suitable substituents include the substituents taught by Kmiec II. Alternative substituents include the substituents taught in U.S. Patent No. 5,334,711 (Sproat) and the substituents taught in Patent Publications EP 629,387 and EP 679,657 (collectively referred to as Martin Applications), which are incorporated herein at as a reference. As used herein, a 2'-fluoro, chloro or bromine derived from a ribonucleotide or a ribonucleotide that has a T-OH substituted with a substituent described in the Martin or Sproat Applications is termed a "T-Substituted Ribonucleotide". As used herein, the term "RNA-like nucleotide" means a T-hydroxyl or 2'-substituted nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the unnatural linkages taught. by Kmiec I or Kmiec II. As used herein, the term "deoxyribo-like nucleotides" means a nucleotide with T-H, which can be linked to other nucleotides of a gene repair oligonucleobase by an unsubstituted phosphodiester linkage or any other unnatural linkages taught by Kmiec I. or Kmiec II.

[0183] In one embodiment of the present disclosure, 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, phosphodiester derivatives and non-phosphorus-based linkages, as taught by Kmiec II. In another embodiment, each RNA-like nucleotide in the mixed duplex oligonucleotide is a 2'-substituted nucleotide. Specifically preferred embodiments of 2'-Substituted Ribonucleotides are 2'-fluoro, T-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, 2'-methoxyethyloxy, T-fluoropropyloxy and 2-substituted ribonucleotides. '-trifluoropropyloxy. 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 phosphodiester linkages.

[0184] Although mixed duplex oligonucleotides (MDONs) with only one type of 2'-substituted RNA-like nucleotide are more conveniently synthesized, the methods of the disclosure can be practiced with mixed duplex oligonucleotides with two or more types of nucleotides of RNA type. The function of a segment of RNA may not be affected by a disruption caused by the introduction of a deoxynucleotide between two RNA-like trinucleotides; accordingly, the term RNA segment encompasses terms such as "interrupted RNA segment". An uninterrupted RNA segment is called a contiguous RNA segment. In an alternative embodiment, an RNA segment may contain alternating 2'-OH unsubstituted and RNase-resistant nucleotides. Mixed duplex oligonucleotides are, in some embodiments, less than 100 nucleotides and, in other embodiments, less than 85 nucleotides but more than 50 nucleotides. The first and second strands have Watson-Crick base parity. In one embodiment, the strands of the mixed duplex oligonucleotide are covalently linked by a linker, such as a single-stranded hexa, penta, or tetranucleotide, such that the first and second strands are single-strand segments of the oligonucleotide with a single 3' end. and a single 5' end. The 3' and 5' ends can be protected by the addition of a "hairpin" termination, whereby the 3' and 5' terminal nucleotides are paired, by Watson-Crick parity, to adjacent nucleotides. A second hairpin-type termination may additionally be positioned at the junction between the first and second strands distant from the 3' and 5' ends, so that the Watson-Crick parity between the first and second strands is stabilized.

[0185] The first and second strands contain regions that are homologous to two fragments of the target gene/allele, that is, they have the same sequence as the target gene/allele. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-like nucleotides from the connecting DNA segment and may also contain DNA-like nucleotides that are not found in the interfering DNA segment. The two regions of homology are separated by, and are each adjacent to, a region with a sequence that differs from the sequence of the target gene, called a "heterologous region." The heterologous region may contain one, two or three incompatible nucleotides. The incompatible nucleotides may be contiguous or, alternatively, they may be separated by one or two nucleotides that are homologous to the target gene/allele. Alternatively, the heterologous region may also contain an insertion or one, two, three, five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the target gene/allele sequence only by deleting one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position of the heterologous region is in this case considered to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are found in the heterologous region. The distance between fragments of the target gene that are complementary to the two homologous regions is identically the length of the heterologous region in which a substitution or substitutions are intended. When the heterologous region contains an insertion, the homologous regions are therefore separated in the mixed duplex oligonucleotide further into the gene/allele than their homologous complementary fragments, and the converse applies when the heterologous region encodes a deletion.

[0186] The RNA segments of the mixed duplex oligonucleotides are each part of a homologous region, that is, a region that is sequentially identical to the target gene fragment, which segments together contain, in some embodiments, at least 13 RNA-like nucleotides and, in some embodiments, 16 to 25 RNA-like nucleotides or even more preferably 18-22 RNA-like nucleotides or, in other embodiments, 20 nucleotides. In one embodiment, RNA segments from regions of homology are separated by and adjacent to, i.e., "connected by" an interfering DNA segment. In one embodiment, each nucleotide of the homologous region is a nucleotide of an interfering DNA segment. A segment of interfering DNA that contains the heterologous region of a mixed duplex oligonucleotide is called a "mutant segment".

[0187] In another embodiment of the present disclosure, the gene repair oligonucleobase (GRON) is a single-stranded oligodeoxynucleotide mutational vector or SSOMV, as disclosed in International Patent Application PCT/USOO/23457, U.S. Patent No. 6,271 360, 6,479,292 and 7,060,500, incorporated by reference in their entirety. The SSOMV sequence is based on the same principles as the mutational vectors described in US Patent No. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Applications No. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789. The SSOMV sequence contains two regions that are homologous to the target sequence separated by a region that contains the desired genetic change called the mutant region. The mutant region may have a sequence with the same length as the sequence that separates the homologous regions in the target sequence, however, having a different sequence. Such a mutant region can generate a substitution. Alternatively, homologous regions in SSOMV may be contiguous with respect to one another, while regions in the target gene that have the same sequence are separated by one, two or more nucleotides. Such SSOMV generates the deletion of the target gene of nucleotides that are absent from SSOMV. Finally, the target gene sequence that is identical to the homologous regions may be adjacent in the target gene but separated by one, two, or more nucleotides in the SSOMV sequence. This SSOMV generates an insertion in the target gene sequence. In some embodiments, an SSOMV does not anneal itself.

[0188] SSOMV nucleotides are deoxyribonucleotides that are linked by unmodified phosphodiester bonds, except that the inter-nucleotide bond at the 3' and/or '5' terminal or, alternatively, the two inter-nucleotide bonds at the terminal '3 and at the '5 terminal, they can be phosphorothioate or phosphoamidate. As used herein, an inter-nucleotide bond is the bond between SSMOV nucleotides and does not include the bond between the 3'-end nucleotide or '5-end nucleotide and a blocking substituent. In a specific embodiment, the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the regions of homology are suitably at least 20 deoxynucleotides in total length and at least two regions of homology must each have lengths of at least 8 deoxynucleotides.

[0189] SSOMV can be designed to be complementary to both the coding and non-coding strands of the target gene. When the desired mutation is a single base substitution, it is preferred that the mutant nucleotide and the target nucleotide are a pyrimidine. To the extent that it is consistent with obtaining the desired functional result, it is preferred that both the mutant nucleotide and the target nucleotide on the complementary strand are pyrimidines. Particularly preferred are SSOMVs that encode transversion mutations, that is, a C or T mutant nucleotide is mismatched, respectively, with a C or T nucleotide on the complementary strand.

[0190] Okazaki/GRON 2'-OME Fragment Projection. In various embodiments, a GRON may have both RNA and DNA nucleotides and/or other types of nucleobases. In some embodiments, one or more of the DNA or RNA nucleotides comprises a modification. In certain embodiments, the first 5' nucleotide is an RNA nucleotide and the rest of the nucleotides are DNA. In still other embodiments, the 5' first RNA nucleotide is modified with a 2-O-Me. In other embodiments, the first two, three, four, five, six, seven, eight, nine, ten or more 5' nucleotides are an RNA nucleotide and the rest of the nucleotides are DNA. In still other embodiments, one or more of the first two, three, four, five, six, seven, eight, nine, ten or more 5' RNA nucleotides are modified with a 2-O-Me. In plant cells, double-strand breaks in DNA are generally repaired via the NHEJ DNA repair pathway. This pathway does not require a template to repair DNA and is therefore prone to errors. The advantage of using this DNA repair pathway for a plant cell is that it is rapid, ubiquitous and, most importantly, can occur at times when a cell is not undergoing DNA replication. Another DNA repair pathway that functions to repair double-strand breaks outside the replication fork in plant cells is called homologous recombination (HR); however, unlike the NHEJ pathway this type of repair is precise and requires the use of a DNA template (GRON). Since these GRONs mimic Okazaki fragments at the DNA replication fork of target genes, it is not obvious to use them with a DNA double-strand cutter for those skilled in the art.

[0191] Improve efficiency

[0192] The present invention provides a number of approaches to increase the conversion efficiency of a target gene, using repair oligonucleotides, and which can be used alone or in combination with each other. They include: 1. Introduction of modifications to the repair oligonucleotides that attract DNA repair machinery to the target site (mismatch). A. Introduction of one or more abasic sites into the oligonucleotide (e.g., within 10 bases, and in some embodiments within 5 bases of the desired mismatch site) generates a lesion that is an intermediate in base excision repair (BER), and which attracts BER machinery to the proximity of the target site for conversion by the repair oligonucleotide. Modified dSpacer (furan abasic) oligonucleotides can be prepared as described in, for example, Takeshita et al., J. Biol. Chem., 262: 10171-79, 1987. B. The inclusion of compounds that induce single or double strand breaks, or in which the oligonucleotide or in conjunction with the oligonucleotide, generates a lesion that is repaired by NHEJ, end joining mediated by microhomology (MMEJ) and homologous recombination. By way of example, the bleomycin family of antibiotics, zinc fingers, FokI (or any type of class IIS restriction enzyme) and other nucleases can be covalently linked to the 3' or 5' end of repair oligonucleotides, to in order to introduce double-strand breaks in the vicinity of the target site for conversion by the repair oligonucleotide. The bleomycin family of antibiotics are DNA-cleaving glycopeptides that include bleomycin, zeocin, phleomycin, talisomycin, pepleomycin, and others. C. Introduction of one or more 8'oxo Da or dG incorporated into the oligonucleotide (e.g., within 10 bases, and in some embodiments within 5 bases of the desired mismatch site) generates a lesion that is similar to the lesions created by reactive species of oxygen. These injuries induce the so-called "push repair" system. See, for example, Kim et al., J. Biochem. Mol. Biol. 37: 657-62, 2004. 2. Increase the stability of repair oligonucleotides: Introduction of a reverse base (IDC) at the 3' end of the oligonucleotide to create a blocked 3' end of the repair oligonucleotide. Introduction of one or more nucleotides or 2'O-methyl bases that increase the hybridization energy (see, for example, WO2007/073149) at the 5' and/or 3' end of the repair oligonucleotide. Introduction of one or a plurality of 2'O-methyl RNA nucleotides at the 5' end of the repair oligonucleotide, which leads to DNA bases that provide the desired mismatch site, thereby creating a DNA fragment-like nucleic acid structure. Okazaki. Conjugated intercalating dyes (5' or 3') such as acridine dyes, psoralen, ethidium bromide and Syber stains. Introduction of a 5' terminal cap, such as T/A clamp, a cholesterol moiety, SIMA (HEX), riboC and amidite. Backbone modifications such as phosphorothioate, 2'-O-methyl, methyl phosphonates, locked nucleic acid (LNA), MOE (methoxyethyl), diPS, and peptide nucleic acid (PNA). Cross-linking the repair oligonucleotide, for example, with intrastrand cross-linking reagents such as cisplatin and mitomycin C. Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5, Cy3B, Cy5 and DY647. 3. Increasing the hybridization energy of the repair oligonucleotide by incorporating bases that increase the hybridization energy (see, for example, WO2007/073149). 4. Increased quality of oligonucleotide repair synthesis using nucleotide multimers (dimers, trimers, tetramers, etc.) as building blocks for synthesis. This results in fewer coupling steps and easier separation of full-length products from building blocks. 5. Use of long repair oligonucleotides (i.e., greater than 55 nucleotides in length, e.g., such as the lengths described herein, e.g., having one or more mutations or two or more mutations targeted by the repair oligonucleotide.

[0193] Examples of the previous approaches are provided in Table1.

[0194] Table 1. Exemplary Gron chemistries.

[0195] The foregoing modifications may also include known nucleotide modifications, such as methylation, 5' intercalating dyes, modifications at the 5' and 3' ends, scaffold modifications, cross-linkers, cyclizations and "caps", and the replacement of one or more nucleotides naturally occurring by an analogue such as inosine. Nucleotide modifications include the addition of acridine, amine, biotin, cascade blue, cholesterol, Cy3@, Cy5@, Cy5.5@ Daboyl, digoxigenin, dinitrophenyl, Edans, 6-FAM, fluorescein, 3'-glyceryl, HEX, IRD-700, IRD-800, JOE, psoralen phosphate, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S", SE, BODIPY°, Marina Blue@, Pacific Blue@, Oregon Green@ , Rhodamine Green@, Rhodamine Red@, Rhodol Green@, and Texas Red@. Polynucleotide support modifications include methylphosphonate, 2'-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2'-OMeRNA. -amino-dA, 2-aminopurine, 3'- (ddA), 3'dA (cordycepin), 7-deaza-dA, 8-Br-dA, 8- oxo-dA, N6-Me-dA, abasic site ( dSpacer), biotin dT, 2'-OMe-5Me-C, 2'-OMe-propynyl-C, 3'- (5-Me- dC), 3'- (ddC), 5-Br-dC, 5- 1-duc, 5-Me-dC,5-F-dC, carboxy-dT, dA convertible, dC convertible, dG convertible, dT convertible, dU convertible, 7-deaza-dG, 8-Br-dG, 8-oxo -dG, O6-Me-dG, S6-DNP-dG, 4-methyl-indole, 5-nitroindole, 2'-OMe-inosine, 2'-dl, o6-phenyl-dl, 4-methyl-indole, 2 '- deoxynobularin, 5-nitroindole, 2-aminopurine, dP (purine analogue), dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT, biotin-dT, carboxy-dT, 04-Me-dT, 04-triazole dT, 2'-OMe-propynyl-U, 5-Br-dU, 2'-dU, 5-F-dU, 5-l-dU, 04-triazole dU. The terms mentioned also cover peptide nucleic acids (PNAs), a DNA analogue in which the support is a pseudopeptide consisting of N-(2-aminoethyl)-glycine units instead of a sugar. PNAs mimic the behavior of DNA and bind complementary nucleic acid strands. The neutral support of PNA results in stronger binding and greater specificity than is typically achieved. Furthermore, the unique chemical, physical and biological properties of PNA have explored the production of powerful biomolecular tools, antisense and antigen agents, molecular probes and biosensors.

[0196] Oligonucleobases can have modified nucleotides with incision(s), gap(s), such as modified oligonucleotide supports, abasic nucleotides and other chemical moieties. In another embodiment, at least one strand of the oligonucleobase includes at least one additional modified nucleotide, for example, a 2'-O-methyl modified nucleotide such as a MOE (methoxyethyl), a nucleotide with a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2-deoxy-2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a blocked nucleotide, an abasic nucleotide (the nucleobase is absent or has a hydroxyl group at the in its place (see, for example, Glen Research, http://www.glenresearch.com/GlenReports/GR21-14.html)), a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidite and a non-natural base comprising a nucleotide. Various salts, mixed salts, and free acid forms are also included.

[0197] Preferable modified oligonucleotide supports include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, 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 with normal 3'-5' linkages, 2'-5' linked analogues thereof and those having reversed polarity in which a or more inter-nucleotide bonds is/are a 3' to 3', 5' to 5' or 2' to 2' bond. Preferred oligonucleotides with inverted polarity comprise a single 3' to 3' inter-nucleotide bond plus 3', i.e., a single inverted nucleotide residue that may be abasic (the nucleobase is absent or has a hydroxyl group in place). this). The most common use of a linkage flip is to add a 3'-3' linkage to the end of an antisense oligonucleotide with a phosphorothioate support. The 3'-3' linkage further stabilizes the antisense oligonucleotide for exonuclease degradation creating an oligonucleotide with two 5'-OH ends and no 3'-OH ends. Bond inversions can be introduced at specific sites during nucleotide oligo synthesis through the use of "inverted phosphoramidites". These reagents have the phosphoramidite groups in the 5'-OH position and the dimethoxytrityl (DMT) protecting group in the 3'-OH position. Typically, the DMT protecting group is on the 5'-OH and the phosphoramidite is on the 3'-OH.

[0198] 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-diaminopurine, 4-thio-uridine, 5-bromo-uridine, 5-fluoro-cytidine, 5-fluorouridine, 5-indo-uridine, 5-methyl-cytidine, inosine, N3-methyl-uridine , 7-deaza-guanine, 8-aminohexyl-amino-adenine, 6-thio-guanine, 4-thio-thymine, 2-thio-thymine, 5-iodo-uridine, 5-iodo-cytidine, 8-bromo-guanine , 8-bromo-adenine, 7-deaza-adenine, 7-diaza-guanine, 8-oxo-guanine, 5,6-dihydro-uridine and 5-hydroxymethyl-uridine. These synthetic units are commercially available (for example, purchased from Glen Research Company) and can be incorporated into DNA by chemical synthesis.

[0199] Examples of modification of the sugar moiety are 3'-deoxylation, 2'-fluorination and arabanosidation, however, it should not be understood as if it were limited to these. The incorporation of these into DNA is also possible by chemical synthesis.

[0200] Examples of 5' end modification are 5'-amination, 5'-biotinylation, 5'-fluoresceylation, 5-tetrafluoro-fluoreceinylation, 5'-thionation and 5'-dabsylation, however they should not be considered limiting this .

[0201] Examples of modification of the 3' end are 3'-amination, 3'-biotinylation, 2,3-dideoxidation, 3'-thionation, 3'-dabsylation, 3'-carboxylation and 3'-cholesterylation, however, should not be considered as limiting this.

[0202] In a preferred embodiment, the oligonucleobase may contain a 5' blocking substituent that is attached to the 5' terminal carbons via a linker. The chemistry of the ligand is not critical, other than its length, which must be, in some embodiments, at least 6 atoms, and the fact that it is flexible. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. In particular, preferable reagents for producing oligonucleobases are reagents marketed as Cy3™ and Cy5™ by GlenResearch, Sterling Va. (now GE Healthcare), which are blocked phosphoramidites that upon incorporation into an oligonucleotide yield 3,3-substituted indomonocarbocyanine. 3',3'-tetramethyl N'-isopropyl and indodicarbocyanine dyes, respectively. Cy3 is particularly preferable. When indocarbocyanine is replaced by N-oxyalkyl, it can be conveniently attached to the 5' end of the oligodeoxynucleotide through a phosphodiester with 5' end phosphate. When commercially available Cy3 phosphoramidite is used as a target, the resulting '5 modification consists of a joint blocking substituent and linker which are an N-hydroxypropyl, N'-phosphatidylpropyl 3,3,3',3'-tetramethyl indomonocarbocyanine. Other dyes contemplated include Rhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Atto550 26, Cy3.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, mStrawberry and mCherry.

[0203] In a preferred embodiment, the indocarbocyanine dye is tetra substituted in the 3 and 3' positions of the indole rings. Without limitations on theory, these substitutions prevent the paint from being an intercalating paint. The identity of the substituents at these positions is not critical.

[0204] The oligo designs described here can also be used as more efficient donor templates in combination with other DNA recombination and editing technologies, including, but not limited to, gene targeting utilizing site-specific homologous recombination by finger nucleases. of zinc, Transcription Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRSs).

[0205] The present disclosure, in some aspects and embodiments, relates, generally, to methods for efficiently modifying cellular genomic DNA and/or recombining DNA into the genomic DNA of cells. Although not limited to any specific use, some methods provided herein may, in some embodiments, be useful in, for example, introducing a modification into a 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 encoding an enzyme to determine whether the modification alters the enzyme's enzymatic activity and/or to determine the location of the enzyme's catalytic region. Alternatively, modification can be introduced into the coding sequence of a DNA-binding protein to determine whether the DNA-binding activity of the protein is altered and thereby delineate the specific DNA-binding region in the protein. Another alternative is to introduce a modification to a non-coding regulatory sequence (e.g., promoter, enhancer, regulatory RNA (miRNA) sequence, etc.) in order to determine the effect of the modification on the expression level of a second sequence that is operationally linked to the non-coding regulatory sequence. This may be desirable to, for example, define the specific sequence that possesses the regulatory activity.

[0206] DNA cutters

[0207] One strategy for producing target gene breaks is through the generation of single-strand breaks or double-strand DNA breaks using a DNA cutter such as a site-specific endonuclease. Endonucleases are most often used for target gene disruption in organisms that have traditionally been refractory to more conventional methods of gene targeting, such as algae, plants, and large animal models, including humans. For example, there are currently human clinical trials involving zinc finger nucleases for the treatment and prevention of HIV infection. Furthermore, endonuclease engineering is currently being used in attempts to disrupt genes that produce undesirable phenotypes in crops.

[0208] Zinc Fingers

[0209] One class of artificial endonucleases are zinc finger endonucleases. Zinc finger endonucleases combine a nonspecific cleavage domain, typically that of Fokl endonuclease, with zinc finger protein domains that are modified to bind to specific DNA sequences. The modular structure of zinc finger endonucleases makes them a versatile platform for delivering site-specific double-strand breaks to the genome. Because the FokI endonuclease cleaves a dimer, one strategy to prevent off-target cleavage events has been to create zinc finger domains that bind to sites 9 base pairs adjacent. See also Pat. U.S. No. 7,285,416.; 7,521,241; 7,361,635; 7,273,923; 7,262,054; 7,220,719; 7,070,934; 7,013,219; 6,979,539; 6,933,113; 6,824,978; each of which is incorporated herein by reference in its entirety.

[0210] TALENs

[0211] TALENS are targetable nucleases used to induce single-strand and double-strand breaks at specific sites in DNA, which are then repaired through mechanisms that can be exploited to create sequence changes at the cleavage site.

[0212] The fundamental building block that is used to design the DNA binding region of TALENs is a highly conserved repeat domain derived from naturally occurring TALEs encoded by proteobacteria Xanthomonas spp. DNA binding by a TALEN is mediated by sets 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.

[0213] These TALE repeats specifically bind to a single DNA base, 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 corresponding to the length of the acid desired target nucleic acid, the identity of the repeat selected to pair with the target nucleic acid sequence. In some embodiments, the target nucleic acid is between 15 and 20 base pairs in length in order to maximize target site selectivity. Cleavage of the target nucleic acid typically occurs within 50 base pairs of TALEN binding. Computer programs for designing TALEN recognition sites have been described in the art. See, for example, Cermak et al., Nucleic Acids Res. 2011 July; 39 (12): E82.

[0214] 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 or administered as mRNA.

[0215] Meganucleases

[0216] 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. Although the specificity of homing endonucleases for their target sites allows precise targeting of induced DNA breaks, homing endonuclease cleavage sites are rare and the probability of finding a naturally occurring cleavage site in a target gene is low.

[0217] Another class of artificial endonucleases is modified meganucleases. Modified homing endonucleases are generated by modifying the specificity of existing homing endonucleases. In one approach, variations are introduced into the naturally occurring amino acid sequence of homing endonucleases and then the resulting engineering product of homing endonucleases is screened to select functional proteins that cleave a target binding site. In another approach, chimeric homing endonucleases are designed by combining the two recognition sites of different homing endonucleases to create a new recognition site consisting of a half-site from each homing endonuclease. See, for example, US Patent No. 8,338,157.

[0218] CRISPRs or CRISPR/cas systems

[0219] CRISPR-Ccas system contains three basic projection components: 1) Cas gene, transcript (e.g., mRNA) or protein; 2) guide RNA (gRNA); and 3) crRNAs (CRISPR RNA) are segments of RNA processed from RNA transcripts encoding CRISPR repeat arrays, which harbor a "protospacer" region that is complementary to a foreign DNA site (e.g., region endogenous DNA target) and a part of the CRISPR repeat. See, for example, PCT Application Nos. WO/2014/093661 and WO/2013/176772.

[0220] Cas Gene (Associated with CRISPR), Transcript (e.g., mRNA) or Protein

[0221] Transient expression of Cas from a plasmid vector, direct delivery of Cas protein and/or direct delivery of Cas mRNA into plant cells. Cas genes are codon-optimized for expression in higher plants, algae or fungi and are driven by a constitutive, inducible, species-specific or tissue-specific promoter, when applicable. Cas polyadenylation and transcript termination signals are either NosT, RBCT, HSP18.2T, or other species- or gene-specific terminators. Cassette gene cassettes or mRNA may contain introns, either natively or in combination with gene-specific promoters and/or synthetic promoters. Cas protein may contain one or more nuclear localization signal (NLS) sequences, mutations, deletions, alterations or truncations. In transient expression systems, cas gene cassettes can be combined with other components of the CRISPRS-cas system, such as gRNA cassettes in the same transient expression vector. Alternatively, Cas gene cassettes can be localized and expressed from independent gRNA cassette constructs or other components of the CRISPR-Cas system. CRISPR-associated gene (CAS) - encodes proteins with a variety of predicted nucleic acid-handling activities, such as nucleases, helicases, and polymerases. Cas genes include cas9. Cas9 is a gene that encodes a large protein containing predicted endonuclease domains similar to RuvC and HNH and is associated with the CRISPRS adaptive immunity system that is present in most archaea and many bacteria. Cas9 protein consists of two lobes: 1) Recognition lobe (REC) - consists of three domains: a) BH (bridge of the helix) b) REC1- facilitates RNA-guided DNA targeting c) REC2- facilitates DNA targeting guided by RNA 2) Nuclease lobe (NUC) - consists of three domains: a) RuvC- facilitates targeting of DNA guided by RNA; endonuclease activity b) HNH - endonuclease activity c) PI-PAM interaction

[0222] In other embodiments, the cas gene may be a homologue of cas9 in which the RuvC, HNH, REC and BH domains are highly conserved. In some embodiments, the cas genes are those of the following species.

[0223] Guide RNA (gRNA)

[0224] gRNA or sgRNA (single guide RNA) was designed as a fusion between a crRNA and part of the CRISPRS transactivation RNA sequence (tracrRNA), which guides Cas9 to a specific target DNA sequence that is complementary to the region of protospacer. Guide RNA may include an expression cassette containing a projection RNA chimeric with a long tracerRNA hybrid, short tracrRNA hybrid, or a native CRISPRS array + tracrRNA conformation. Chimeric gRNA combines the targeting specificity of crRNA with the scaffolding properties of tracrRNA into a single transcript. Transient gRNA expression is controlled by species-specific higher plant RNA Polymerase III promoters, such as those of the U3 or U6 gene or snRNA family (Wang et al 2008). Termination of the gRNA transcript is controlled by a 6-20 nucleotide poly dT tract as per Wang et al 2008. The gRNA expression cassettes are located in the same or different vectors for transient expression of other components of the CRISPRS-cas system. . gRNA transcripts can be synthesized in vitro and delivered directly into plant cells, independently of or in combination with transient gRNA expression vectors.

[0225] Target Region

[0226] Guide RNAs contain two components that define specificity for a DNA target region, a proto-spacer and a proto-spacer adjacent motif (PAM). Protospacer sequence, typically 20 nucleotides long but may vary depending on the DNA target, provides DNA sequence specificity for the CRISPR-Cas complex. DNA targets also contain a NAG or NNG tri-nucleotide sequence (PAM) where N indicates any nucleotide immediately 3' or downstream of the protospacer.

[0227] One-component approach

[0228] Similar to Le Cong et al. (2013) and others, a simplified "one-component approach" to CRISPRS-cas gene editing, in which a single transient expression construct contains all components of the CRISPR-Cas complex, i.e., both the gRNA expression cassettes how much of Cas. This allows for easy modular design for targeting single or multiple loci in any plant or crop. Targeting multiple loci can be achieved by simply swapping target-specific gRNA cassettes. Furthermore, species-specific promoters, terminators, or other expressing enhancer elements can easily be pushed in and out of transient "one-component approach" vectors that allow for optimal expression of both gRNA and Cas proteins in a species specific.

[0229] Two-Component Approach

[0230] In the two-component approach, Cas and gRNA expression cassettes are located in different transient expression vectors. This allows the delivery of an edited CRISPR-Cas component separately, allowing for different ratios of gRNA to Cas within the same cell. Similar to the one-component approach, the two-component approach also allows promoters, terminators, or other elements that affect the expression of CRISPR-Cas components to be easily altered and allows targeting of DNA in a species-specific manner.

[0231] Antibiotics

[0232] Another class of endonucleases are antibiotics that are DNA-cleaving glycopeptides, such as the bleomycin family of antibiotics are DNA-cleaving glycopeptides that include bleomycin, zeocin, phleomycin, talisomycin, pepleomycin and others that are further described herein .

[0233] Other DNA modifying molecules can be used in targeted gene recombination. For example, peptide nucleic acids can be used to induce modifications in the genome of the target cell or cells (per, e.g., Ecker, U.S. Patent No. 5,986,053, to, incorporated herein by reference). In summary, synthetic nucleotides comprising at least a partial peptide support are used to target a homologous genomic nucleotide sequence. Upon binding to double-stranded DNA, or through a mutagen linked to the peptide nucleic acid, modification of the target DNA sequence and/or recombination is/are induced to occur. Targeting specificity is determined by the degree of sequence homology between the targeting sequence and the genomic sequence.

[0234] Furthermore, the present disclosure is not limited to the specific methods that are used here to perform modification of genomic sequences. In fact, a series of methods are contemplated. For example, genes can be targeted using triple helix forming oligonucleotides (TFO). TFOs can be generated synthetically, for example, by PCR or by the use of a gene synthesizing apparatus. Additionally, TFOs can be isolated from genomic DNA if appropriate natural sequences are found. TFOs can be used in a variety of ways, including, for example, by binding to a mutagen such as, but not limited to, a psoralen or chlorambucil (see, e.g., Havre et al., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., JVirol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995; , U.S.A. 88:5602-5606, 1991; Belousov et al., Nucleic Acids Res 25:34403444, 1997). Furthermore, for example, TFOs can be joined to donor duplex DNA (see, for example, Chan et al., J Biol Chem272:11541-11548, 1999). TFOs can also act by binding with enough affinity to trigger error-prone repair (Wang et al., Science 271:802-805, 1996).

[0235] The methods disclosed herein are not necessarily limited to the nature or type of the DNA modifying reagent. For example, such DNA-modifying reagents release radicals that result in DNA strand breakage. Alternatively, the reagents alkalize DNA to form adducts that would block replication and transcription. In another alternative, the reagents generate cross-links or molecules that inhibit cellular enzymes leading to strand breaks. Examples of DNA modifying reagents that have been linked to oligonucleotides to form TFOs include, but are not limited to, indolocarbazoles, naphthalene diimide (NDI), transplatin, bleomycin, cyclopropapyrroloindole analogs, and phenanthodihydrodiozines. Specifically, indolocarbazoles are inhibitors of topoisomerase I. Inhibition of these enzymes results in strand breaks and formation of DNA protein adducts [Arimondo et al., Bioorganic and Medicinal Chem. 8, 777, 2000]. NDI is a photo-oxidant that can oxidize guanines that could cause mutations at guanine residue sites [Nunez, et al., Biochemistry, 39, 6190, 2000]. Transplatin, as has already been demonstrated, reacts with DNA in a triplex target when the TFO is linked to the reagent. This reaction causes the formation of DNA adducts that would be mutagenic [Columbier, et al., Nucleic Acids Research, 24: 4519, 1996]. Bleomycin is a DNA switch, widely used as a radiation mimetic. It has been linked to oligonucleotides and has been shown to be active as a switch in this format [Sergeyev, Nucleic Acids Research 23, 4400, 1995; Kane, et al., Biochemistry, 34, 16715, 1995]. Cyclopropapyrroloindole analogs have been linked to TFOs and have been shown to alkalize DNA at a target triplex sequence. The alkylated DNA would then contain adducts that 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 associated with TFOs and have been shown to introduce breaks in duplex DNA in the event of photoactivation [Bendinskaset al., Bioconjugate Chem. 9, 555, 1998].

[0236] Other methods of inducing modifications and/or recombination are contemplated by the present disclosure. For example, another modality involves the induction 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 the use of peptide nucleic acids (PNA ) with affinity for the targeted site. Still other methods include specific DNA recognition and targeting by polyamides (see, e.g., Dervan et al., Curr Opin Chem Biol 3:688-693, 1999; Biochemistry 38:2143-2151, 1999) and the use of nucleases with site-specific activity (e.g. zinc finger proteins, TALENs, meganucleases and/or CRISPRs).

[0237] The present disclosure is not limited to any specific frequency of modification and/or recombination. In some embodiments, the methods disclosed herein result in a frequency of modification in the target nucleotide sequence of 0.2% to 3%. Nevertheless, any frequency (i.e., between 0% and 100%) of modification and/or recombination is contemplated to be within the scope of the present disclosure. The frequency of modification and/or recombination depends on the method used to induce the modification and/or recombination, the cell type used, the specific gene targeted, and the DNA mutation reagent used, if any. Additionally, the method used to detect modification and/or recombination, due to limitations in the detection method, may not detect all instances of modification and/or recombination. Furthermore, some modification and/or recombination events may be silent, providing no detectable indication that modification and/or recombination has occurred. The inability to detect silent modification and/or recombination events provides an artificially low estimate of modification and/or recombination. For these reasons, among others, disclosure is not necessarily limited to any specific frequency of modification and/or recombination. In one embodiment, the frequency of modification and/or recombination is between 0.01% and 100%. In another embodiment, the frequency of modification and/or recombination is between 0.01% and 50%. In yet another modality, the frequency of modification and/or recombination is between 0.1% and 100%. In yet another modality, the frequency of modification and/or recombination is between 0.1% and 5%.

[0238] The term "mutation frequency", as used herein, in reference to a population of cells that are treated with a DNA modifying molecule that is capable of introducing mutation at a target site in the cells' genome, refers to a number of cells in the treated population that contain the mutation at the target site compared to the total number of cells treated with the DNA modifying molecule. For example, for a population of cells treated with the psoralen-linked DNA-modifying molecule TFO that is designed to induce a mutation at a targeted location in the cell's genome, a mutation frequency of 5% means that A total of 100 cells that are treated with TFO-psoralen, 5 cells contain a target site mutation.

[0239] Although the present disclosure is not necessarily 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 disclosure require higher degrees of precision, depending on the desired result. For example, the specific sequence changes required for gene repair (e.g., specific base changes) require a greater degree of precision compared to producing a gene knockout where only gene disruption is required. With the methods of the present disclosure, obtaining higher levels of precision in modification and/or homologous recombination techniques is superior to that of prior art methods.

[0240] Distribution of oligonucleobase repair gene in plant cells

[0241] Any known method currently used to transform a plant cell can be used to deliver the gene repair oligonucleobases. Illustrative methods are listed below. The methods and compositions described herein may involve any of many methods for transfecting cells with the DNA modifying reagent or reagents. Methods for introducing DNA modifying reagents into a cell or cells are well known in the art and include, but are not limited to, microinjection, electroporation, passive adsorption, calcium phosphate-DNA coprecipitation, DEAE-dextran mediated transfection, polybrene-mediated, liposome fusion, lipofectin, nucleofection, protoplast fusion, retroviral infection, biolistic (i.e. particle bombardment) and the like.

[0242] The use of metallic microcarriers (microspheres) for the introduction of large DNA fragments into plant cells with cellulose cell walls by means of projectile penetration is known to individuals skilled in the relevant art (hereinafter biolistic administration). US Patent No. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microchargers and devices for designing them.

[0243] Specific conditions for the use of microchargers in the methods disclosed herein may include the conditions described in International Publication WO 99/07865. In an illustrative technique, ice-cold microcarriers (60 mg/ml), mixed duplex oligonucleotides (60 mg/ml), 2.5 M CaCl2, and 0.1 M spermidine are added in this order; the mixture is gently stirred, for example by means of a vortex, for 10 minutes, and allowed to settle at room temperature for 10 minutes, when the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol . Good results can be obtained with a concentration in the microcarrier adhesion solution of 8-10 μg/μl, 14-17 μg/ml mixed duplex oligonucleotides, 1.1-1.4 M CaCl2 and 18-22 mM spermidine. Optimal results were observed under conditions of 8 μg/μL microcarriers, 16.5 μg/mL mixed duplex oligonucleotide, 1.3 M CaCl2, and 21 mM spermidine.

[0244] Gene repair oligonucleobases can also be introduced into plant cells using microfibers to penetrate the cell wall and cell membrane. US Patent No. 5,302,523 to Coffee et al. describe the use of silicon carbide fibers to facilitate the transformation of corn crops into Black Mexican Sweet suspension. Any mechanical technique that can be used to introduce DNA into the transformation of a plant cell using microfibers that can be used for delivery of gene repair oligonucleobases for transmutation.

[0245] An illustrative technique for microfiber delivery of a gene repair oligonucleobase is as follows: Sterile microfibers (2 μg) are suspended in 150 μl of plant culture medium containing about 10 μg of an oligonucleotide mixed duplex. A suspension culture is allowed to settle and equal volumes of pooled 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 hours, as is appropriate for the specific trait.

[0246] In an alternative embodiment, gene repair oligonucleobases can be delivered to the plant cell by means of electroporation of a protoplast derived from a plant part according to techniques that are well known to an individual moderately skilled in the art. Protoplasts are formed by enzymatic treatment of a part of the plant, particularly a leaf, according to techniques well known to those skilled in the art. See, for example, Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J. Protoplasts do not need to be grown in culture media before electroporation. Illustrative conditions for electroporation are 300,000 protoplasts in a total volume of 0.3 mL with a gene repair oligonucleobase concentration between 0.6-4 μg/mL.

[0247] In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the modifying agent polyethylene glycol, according to techniques well known to those skilled in the art. In another alternative embodiment, gene repair oligonucleobases can be delivered by injecting with a microcapillary into plant cells or protoplasts.

[0248] In an alternative embodiment, nucleic acids are fixed in microspheres composed of calcium alignate and taken up by plant protoplasts in the presence of the membrane modifying agent polyethylene glycol (see, for example, Sone et al., 2002 , Liu et al., 2004).

[0249] In an alternative embodiment, nucleic acids are frozen in water and introduced into plant cells by bombardment in the form of microparticles see, for example, Gilmore, 1991, U.S. Patent 5,219,746; Brinegar et al.)

[0250] In one embodiment, nanoparticle-coupled nucleic acids are introduced into intact plant cells by incubating the cells in a suspension containing the nanoparticles (see, e.g., Pasupathy et al., 2008) or by delivering them into intact cells through from particle bombardment or in protoplasts by co-incubation (see, e.g., Torneyet al., 2007).

[0251] In an alternative embodiment, nucleic acids are complexed with penetration peptides and distributed into cells by co-incubation (see, for example, Chugh et al., 2008, WO 2008148223 A1; Eudes and Chugh.

[0252] In an alternative embodiment, nucleic acids are introduced into intact cells through electroporation (see, for example, He et al., 1998, US 2003/0115641 Al, Dobres et al.)

[0253] In an alternative embodiment, nucleic acids are delivered into dried embryo cells by immersion in a solution with nucleic acids (see, for example, Topfer et al., 1989, Senaratna et al., 1991) or in other embodiments are introduced by Cellsqueeze (SQZ Biotech),

[0254] Vegetable selection

[0255] In various embodiments, plants such as those disclosed herein may be any species of dicotyledonous, monocotyledonous or gymnospermic plant, including any woody plant that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruit, seeds or vegetables, or any species that produces colorful or aromatic flowers. By way of example, the plant may be selected, perhaps, from a plant species of the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum , tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, soybean, soybean spp, sugarcane, pea, chickpea, pea, fava bean, lentils, turnip , Brussels sprouts, lupins, cauliflower, kale, favela, poplar, pine, eucalyptus, grapes, citrus, triticale, alfalfa, rye, oats, grass and forage grasses, flax, rapeseed oil, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris and lily, and nut bearing plants to the extent not specifically mentioned heretofore.

[0256] Plants and plant cells can be tested to verify their resistance and tolerance to the herbicide using methods known in the art, for example by cultivating the plant or plant cell in the presence of a herbicide and measuring the rate of growth velocity compared to the growth rate in the absence of the herbicide.

[0257] As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a rate of growth or rate of cell division of the plant, plant organ, plant tissue or plant cell that is at least at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or cell division rate in a plant, plant organ, plant tissue or corresponding plant cell that expresses the relevant wild-type protein.

[0258] 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 developmental events in the plant, plant organ, plant tissue or plant cell that are substantially identical to those occurring in a corresponding plant, plant organ, plant tissue, or plant cell expressing a wild-type protein.

[0259] In certain embodiments, plant organs provided herein include, but are not limited to, leaves, stems, roots, vegetative shoots, flower buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits or sections, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, soil tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, vesicular tissues, plant tumor tissues, and reproductive tissues . Plant cells include, but are not limited to, isolated cells with cell walls, aggregates thereof of varying dimensions, and protoplasts.

[0260] Plants are substantially "tolerant" to a relevant herbicide when they are subjected to it and provide a dose/reaction curve that is shifted to the right compared to that provided by a similar non-tolerant plant similarly subjected. Such doses/response curves have dose represented on the Plants that are substantially resistant to herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to herbicide at concentrations and rates that are typically employed by the agrochemical community to kill undesirable plants in the field. Plants that are resistant to an herbicide are also herbicide tolerant.

[0261] Vegetable generation

[0262] Tissue culture of various tissues of plant species and regeneration of plants therefrom is known. For example, propagation of a canola cultivar by tissue culture is described in any of the following, but not limited to any of them: Chuong et al., "A Simple Culture Method for Brassica hypocotyls Protoplasts," Plant Cell Reports 4: 4-6, 1985; Barsby, T. L., et al., "A Rapid and Efficient Alternative Procedure for the Regeneration of Plants from Hypocotyl Protoplasts of Brassica napus," Plant Cell Reports (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 Explants of Brassicas," Plant Cell Reports (Spring 1988); Swanson, E., "Microspore Culture in Brassica," Methods in Molecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

[0263] Further reproduction of the variety can occur through tissue culture and regeneration. Tissue culture of various soybean tissues and regeneration of plants therefrom is well known in the art and widely published. For example, reference may be made to Komatsuda, T. et al, "Genotype Stephens, P. A., et al., "Agronomic Evaluation of Tissue-Culture-Derived Soybean Plants," Theor. Appl. Genet. 82:633-635, 1991; Komatsuda, T. et al., "Maturation and Germination of Somatic Embryos as Affected by Sucrose and Plant Growth Regulators in Soybeans Glycine gracilis Skvortz 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 (Glycine max L. Merr.); 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 Organogenesis in Glycine max (Merrill.) by Allantoin and Amides," Plant Science 81:245-251, 1992. The disclosures of US Patent No. 5,024. 944, issued June 18, 1991 to Collins et al., and US Patent No. 5,008,200, issued April 16, 1991 to Ranch, et al, are incorporated herein in their entirety by way of reference.

[0264] Exemplary Modalities

[0265] In addition to the aspects and embodiments described and provided elsewhere in this description, the following non-limiting list of particular embodiments is specifically contemplated. 1. A method for causing a genetic change in a plant cell, said method comprising exposing said cell to a DNA cutter and a modified GRON. 2. A cell comprising a DNA cutter and a GRON. 3. The method or cell of any of the preceding embodiments, wherein said cells are one or more species of cells selected from the group consisting of plants, bacteria, yeast, fungi, algae and mammals. 4. The method or cell of any of the preceding embodiments, wherein said cells are one or more species of cells selected from the group consisting of Escherichia coli, Mycobacterium smegmatis, Baccillus subtilis, Chlorella, Bacillus thuringiensis, Saccharomyces cerevisiae, Yarrowia lipolytica, Chlamydamonas rhienhardtii, Pichia pastoris, Corynebacterium, Aspergillus niger and Neurospora crassa. Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Glycine max, Amaranthus tuberculatus, Linum usitatissimum, and Zea mays 5. The method or cell of any of the preceding embodiments, wherein said cell is Yarrowia lipolytica. 6. The method or cell of any of the preceding embodiments, wherein said cell is a yeast cell other than Saccharomyces cerevisiae. 7. A method for causing a genetic change in a plant cell, said method comprising exposing said cell to a DNA cutter and a modified GRON. 8. A plant cell comprising a DNA cutter and a modified GRON. 9. A method for causing a genetic change in a plant cell, said method exposing said cell to a DNA cutter and a GRON comprising DNA and RNA. 10. A plant cell characterized by the fact that it comprises a DNA cutter comprising DNA and RNA. 11. A method of causing a genetic change in an acetyl coenzyme A carboxylase (ACCase) gene in a cell, wherein said genetic modification causes a change in the Acetyl coenzyme A carboxylase (ACCase) protein in one or plus amino acid positions, said positions selected from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on foxtail reference sequence numbering SEQ ID NO: 1 or a residue of analogous amino acid in an ACCase paralog, said method comprising exposing said cell to a modified GRON. 12. A method of causing a genetic change in an acetyl coenzyme A carboxylase (ACCase) gene in a cell, wherein said genetic modification causes a change in the Acetyl coenzyme A carboxylase (ACCase) protein in one or further amino acid positions, said positions selected from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on foxtail reference sequence numbering SEQ ID NO: 1 or a residue of analogous amino acid in an ACCase paralog, said method comprising exposing said cell to a DNA cutter and a modified GRON. 13. A method for producing a plant or plant cell, which comprises introducing into a plant cell a gene repair oligonucleobase (GRON) with a target mutation of an acetyl coenzyme A carboxylase (ACCase) gene to produce a cell of a plant having an ACCase gene that expresses an ACCase protein comprising a mutation at the positions of one or more amino acids corresponding to a position selected from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based in foxtail reference sequence numbering SEQ ID NO: 1 or in an analogous amino acid residue in an ACCase paralog. 14. A method for producing a plant cell or plant, which comprises introducing into a plant cell a DNA cutter and a gene repair oligonucleobase (GRON) with a target mutation of an acetyl coenzyme A carboxylase gene ( ACCase) for producing a plant cell with an ACCase gene that expresses an ACCase protein comprising a mutation in one or more amino acid positions corresponding to a position selected from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on the numbering of the foxtail reference sequence SEQ ID NO: 1 or an amino acid residue in a paralogous analogue of ACCase. 15. A fertile plant comprising an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation at position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or a analogous amino acid residue in an ACCase paralog. 16. A fertile rice plant comprising an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation at position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog. 17. A plant cell comprising an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation at position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog and further comprising an acetyl coenzyme carboxylase (ACCase) gene encoding a protein comprising a mutation at one or more amino acid positions, said positions selected from the group consisting of 1781, 1783, 1786, 2079, 2080 and 2088 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog. 18. A fertile plant comprising an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation at position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or a analogous amino acid residue in an ACCase paralog and further comprising an acetyl coenzyme carboxylase (ACCase) gene encoding a protein comprising a mutation in one or more amino acid positions, said positions selected from the group consisting of 1781, 1783 , 1786, 2079, 2080 and 2088 based on the numbering of the foxtail reference sequence SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog. 19. A method of causing a genetic change in an Acetyl Coenzyme A Carboxylase (ACCase) gene in a cell, wherein said genetic modification causes a change in the Acetyl Coenzyme A Carboxylase (ACCase) of the protein at a position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog, said method comprising exposing said cell to a modified GRON. 20. A method of causing a genetic change in an Acetyl Coenzyme A Carboxylase (ACCase) gene in a cell, wherein said genetic modification causes a change in the Acetyl Coenzyme A Carboxylase (ACCase) protein at a position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog, said method comprising exposing said cell to a DNA cutter and a modified GRON. 21. The method, plant or cell of any of the foregoing embodiments, wherein said mutation or change in an Acetyl-Coenzyme A carboxylase (ACCase) gene, if present, results in an Acetyl-Coenzyme A protein carboxylase (ACCase) comprising one or more selected from the group consisting of an isoleucine to alanine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for leucine at a position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for methionine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for asparagine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for serine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine to threonine at a position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for valine at the position corresponding to position 1781 of SEQ ID NO: 1; a glycine to cysteine at the position corresponding to position 1783 of SEQ ID NO: 1; an alanine for proline at a position corresponding to position 1786 of SEQ ID NO: 1; an aspartate for glycine at the position corresponding to position 2078 of SEQ ID NO: 1; an aspartate to lysine at a position corresponding to position 2078 of SEQ ID NO: 1; an aspartate to threonine at a position corresponding to position 2078 of SEQ ID NO: 1; a serine for phenylalanine at a position corresponding to position 2079 of SEQ ID NO: 1; a lysine for glutamate at a position corresponding to position 2080 of SEQ ID NO: 1; a cysteine for phenylalanine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for glycine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to histidine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to lysine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for leucine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for asparagine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for proline at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for glutamine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to arginine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to serine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to threonine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to valine at the position corresponding to position 2088 of SEQ ID NO: 1; and a cysteine for tryptophan at the position corresponding to position 2088 of SEQ ID NO: 1. 22. The plant or cell of any of the preceding embodiments, or a plant or a plant cell, made by any of the methods of the preceding embodiments, wherein said plant or cell comprises an Acetyl-Coenzyme A carboxylase (ACCase) protein comprising one or more selected from the group consisting of an isoleucine to alanine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for leucine at a position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for methionine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for asparagine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for serine at the position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine to threonine at a position corresponding to position 1781 of SEQ ID NO: 1; an isoleucine for valine at the position corresponding to position 1781 of SEQ ID NO: 1; a glycine to cysteine at the position corresponding to position 1783 of SEQ ID NO: 1; an alanine for proline at a position corresponding to position 1786 of SEQ ID NO: 1; an aspartate for glycine at the position corresponding to position 2078 of SEQ ID NO: 1; an aspartate to lysine at a position corresponding to position 2078 of SEQ ID NO: 1; an aspartate to threonine at a position corresponding to position 2078 of SEQ ID NO: 1; a serine for phenylalanine at a position corresponding to position 2079 of SEQ ID NO: 1; a lysine for glutamate at a position corresponding to position 2080 of SEQ ID NO: 1; a cysteine for phenylalanine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for glycine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to histidine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to lysine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for leucine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for asparagine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for proline at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine for glutamine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to arginine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to serine at the position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to threonine at a position corresponding to position 2088 of SEQ ID NO: 1; a cysteine to valine at the position corresponding to position 2088 of SEQ ID NO: 1; and a cysteine for tryptophan at the position corresponding to position 2088 of SEQ ID NO: 1. 23. The plant or cell of any of the previous embodiments, or a plant or cell made by any of the methods of the previous embodiments, wherein said plant or plant cell comprises an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation in one or more amino acid positions, said positions selected from the group consisting of 1781, 1783, 1786, 2078, 2079 , 2080 and 2088 based on foxtail reference sequence numbering SEQ ID NO: 1 or to an analogous amino acid residue in an ACCase paralog. 24. The plant or cell of any of the preceding embodiments, or a plant or cell made by any of the methods of the preceding embodiments, wherein said plant or cell comprises an acetyl coenzyme A carboxylase (ACCase) gene encoding a protein comprising a mutation at position 2078 based on foxtail reference sequence numbering SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog and further comprising an acetyl coenzyme A carboxylase gene ( ACCase) encoding a protein comprising a mutation at one or more amino acid positions, said positions selected from the group consisting of 1781, 1783, 1786, 2079, 2080 and 2088 based on the numbering of the tail-tail reference sequence -fox SEQ ID NO: 1 or an analogous amino acid residue in an ACCase paralog.

[0266] In each of the foregoing embodiments of ACCase 1124, whether the methods, plants, cells, or otherwise, the following are suitable mutations for use therein:

[0267] Alternative mutations include, but are not limited to, the following:

[0268] With regard to embodiments 11-24, positions corresponding to 1781m 1783, 1786, 2078, 2079, and 2080 based on foxtail reference sequence numbering are well known in the art and can be readily obtained from appropriate sequence databases. As an example, the following table shows the corresponding positions in the rice ACCase sequence: Am: Alopecurus myosuroid; OsI: Oryza sativa of indica variety; OsJ: Oryza sativa variety japonica 25. A method for producing a plant or plant cell with a mutated EPSPS gene, which comprises introducing into a plant cell a gene repair oligonucleobase (GRON) with a target mutation of a gene 5-enol pyruvylxykimate-3-phosphatase synthase (EPSPS) to produce a plant cell with an EPSPS gene that expresses an EPSPS protein comprising a mutation in the positions of one or more amino acids corresponding to a position selected from the group that consists of 96, 97 and 101 based on amino acid sequence numbering for the Escherichia coli reference sequence SEQ ID NO: 2 or an analogous amino acid residue in an EPSPS paralog. 26. A method for producing a plant or plant cell with a mutated EPSPS gene, which comprises introducing into a plant cell a DNA cutter and a gene repair oligonucleobase (GRON) with a target mutation of a 5- enol pyruvylxikimate-3-phosphatase synthase (EPSPS) to produce a plant cell with an EPSPS gene that expresses an EPSPS protein comprising a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of 96, 97 and 101 based on amino acid sequence numbering for the Escherichia coli reference sequence SEQ ID NO: 2 or an analogous amino acid residue in an EPSPS paralog. 27. A plant or cell with a mutated EPSPS gene, wherein said plant or cell is made by a method of introducing into a plant cell a DNA cutter and a gene repair oligonucleobase (GRON) with a target mutation in a 5-enol pyruvylxykimate-3-phosphatase synthase (EPSPS) gene to produce a plant cell with an EPSPS gene that expresses an EPSPS protein comprising a mutation at one or more amino acid positions corresponding to a position selected from the group that consists of 96, 97 and 101 based on amino acid sequence numbering for the Escherichia coli reference sequence SEQ ID NO: 2 or an analogous amino acid residue in an EPSPS paralog. 28. The plant or cell of any of the preceding embodiments, or a plant or cell made by any of the methods of the preceding embodiments, wherein the plant cell or plant expresses an EPSPS protein comprising a mutation in one or more amino acid positions are selected from the group consisting of a glycine to alanine in the position corresponding to position 96 of SEQ ID NO: 2; a threonine for isoleucine at the position corresponding to position 97 of SEQ ID NO: 2; a proline to alanine at the position corresponding to position 101 of SEQ ID NO: 2; a proline to serine at the position corresponding to position 101 of SEQ ID NO: 2; and a proline to threonine at the position corresponding to position 101 of SEQ ID NO: 2. 29. The plant or cell of any of the preceding embodiments, or a plant or cell made by any of the methods of the preceding embodiments, wherein the cell vegetable or plant expresses an EPSPS protein comprising mutation combinations selected from the group consisting of a threonine to isoleucine at a position corresponding to position 97 of SEQ ID NO: 2 and a proline to alanine at a position corresponding to position 101 of SEQ ID NO: : two; a threonine for isoleucine in the position corresponding to position 97 of SEQ ID NO: 2 and a proline for alanine in the position corresponding to position 101 of SEQ ID NO: 2; a threonine for isoleucine in the position corresponding to position 97 of SEQ ID NO: 2 and a proline for serine in the position corresponding to position 101 of SEQ ID NO: 2; and a threonine for isoleucine in the position corresponding to position 97 of SEQ ID NO: 2 and a proline for threonine in the position corresponding to position 101 of SEQ ID NO: 2.

[0269] With regard to embodiments 25-30, positions corresponding to 96, 97 and 101 based on the numbering of the Escherichia coli reference sequence SEQ ID NO: 2 are well known in the art and easily obtained from databases. appropriate sequence data. See, for example, US Patent No. 8,268,622. As an example, the following table shows the corresponding positions in the linen EPSPS sequence:

[0270] E. coli EPSPS sequence is AroA having the sequence MESLTLQPIARVDGTINLPGSKTVSNRALLLAALAHGKTVLTNLLDSDDVRH MLNALTALGVSYTLSADRTRCEIIGNGGPLHAEGALELFLGNAGTAMRPLAA ALCLGSNDIVLTGEPRMKERPIGHLVDALRLGGAKITYLEQENYPPLRLQGG FTGGNVDVDGSVSSQFLTALLMTAPL APEDTVIRIKGDLVSKPYIDITLNLMK TFGVEIENQHYQQFVVKGGQSYQSPGTYLVEGDASSASYFLAAAAIKGGTV KVTGIGRNSMQGDIRFADVLEKMGATICWGDDYISCTRGELNAIDMDMNHI PDAAMTIATAALFAKGTTRLRNIYNWRVKETDRLFAMATELRKVGAEVEEG HDYIRITPPEKLNFAEIATYND HRMAMCFSLVALSDTPVTILDPKCTAKTFPD YFEQLARISQAA

[0271] Flax 1 gene sequence is LCL - g41452_1333 having the sequence MALVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRRSGNVAA AAAAPLRVSASLTTAAEKASTVPEEVVLQPIKDISGIVTLPGSKSLSNRILLLAALS EGTTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGCGG VFPVGKLAK NDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQLG ADVTCSSTSCPPVHVNGQGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEIEI VDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEGDA SSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEV LEKMGAKVIWTENSVTVT GPPRDASGRKHLRAVDVNMNKMPDVAMTLAVVALYADGPTAIRDVASWRVKE TERMIAICTELRKLGATVEEGPDYCIITPPEKLNIAEIDTYDDHRMAMAFSLAACA DVPPVTIRDPGCTKKTFPDYFEVLERYTKH

[0272] Flax 2 gene sequence is LCL - g40547_1271 having the sequence MAQVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRLLGNVAA AAAAAPLRISASLATAAEKASTVPEEIVLQPIKDISGIVTLPGSKSLSNRILLLAALS EGKTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGRGGVFP VGKLG KNDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQL GADVSCSSTSCPPVHVNAKGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEI EIVDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEG DASSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEVLEK 30. The method or cell according to any of the preceding embodiments, wherein said DNA cutter is a or more selected from a CRISPRS, a TALEN, a zinc finger, meganuclease and a DNA-cleaving antibiotic. 31. The method or cell, according to any of the previous embodiments, wherein said DNA cutter is a CRISPRS or a TALEN. 32. The method or cell, according to any of the preceding embodiments, wherein said DNA cutter is a CRISPRS. 33. The method or cell, according to any of the preceding embodiments, wherein said DNA cutter is a TALEN. 34. The method or cell according to any of the preceding embodiments, wherein said DNA cutter is one or more of the DNA-cleaving antibiotics selected from the group consisting of bleomycin, zeocin, phleomycin, talisomycin and pepleomycin . 35. The method or cell, according to any of the preceding embodiments, wherein said DNA cutter is zeocin. 36. The method or cell, according to any of the previous embodiments, wherein said GRON is single-stranded. 37. The method or cell, according to any of the preceding embodiments, wherein the GRON is a chemically protected oligonucleotide. 38. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises an oligonucleotide chemically protected at the 5' end. 39. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises an oligonucleotide chemically protected at the 3' end. 40. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises an oligonucleotide chemically protected at the 3' and 5' end. 41. Method, according to any of the previous embodiments, wherein the GRON comprises one or more selected from a Cy3 group, a 3PS group and a 2'-O-methyl group. 42. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises a Cy3 group. 43. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a Cy3 group in the first base at the 5' end. 44. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a Cy3 group in the first base at the 3' end. 45. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises a 3PS group. 46. The method or cell, according to any of the previous embodiments, wherein the GRON comprises two or more 3PS groups. 47. The method or cell, according to any of the previous embodiments, wherein the GRON comprises three or more 3PS groups. 48. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 3PS group in the first base at the 5' end. 49. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 3PS group in the first base at the 3' end. 50. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group. 51. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises two or more 2'-O-methyl groups. 52. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in the first base at the 5' end. 53. The method or cell, according to any of the preceding embodiments, wherein the GRON has a 2'-O-methyl group in the first base at the 5' end and has no other 2'-O-methyl groups. 54. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first two or more bases at the 5' end. 55. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first three or more bases at the 5' end. 56. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first four or more bases at the 5' end. 57. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first five or more bases at the 5' end. 58. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first six or more bases at the 5' end. 59. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first seven or more bases at the 5' end. 60. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first eight or more bases at the 5' end. 61. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first nine or more bases at the 5' end. 62. The method or cell according to any of the preceding embodiments, wherein the GRON comprises a 2'-O-methyl group in each of the first ten or more bases at the 5' end. 63. The method or cell, according to any of the preceding embodiments, wherein the GRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 RNA bases at the 5' end. 64. The method or cell, according to any of the previous embodiments, wherein said GRON has an oscillating base pair relative to the target sequence for genetic alteration. 65. The method or cell, according to any of the previous embodiments, wherein said GRON is between 15 and 60 nucleotides in length. 66. The method or cell, according to any of the previous embodiments, wherein said GRON is 41 nucleotides in length. 67. The method or cell, according to any of the previous embodiments, wherein said GRON is between 50 and 110 nucleotides in length. 68. The method or cell, according to any of the previous embodiments, wherein said GRON is 101 nucleotides in length. 69. The method or cell, according to any of the previous embodiments, wherein said GRON is between 150 and 210 nucleotides in length. 70. The method or cell, according to any of the previous embodiments, wherein said GRON is 201 nucleotides in length. 71. The method or cell, according to any of the previous embodiments, wherein said GRON is between 70 and 210 nucleotides in length. 72. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 70 nucleotides in length. 73. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 100 nucleotides in length. 74. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 165 nucleotides in length. 75. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 175 nucleotides in length. 76. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 185 nucleotides in length. 77. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 195 nucleotides in length. 78. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 200 nucleotides in length. 79. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 210 nucleotides in length. 80. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 220 nucleotides in length. 81. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 230 nucleotides in length. 82. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 240 nucleotides in length. 83. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 250 nucleotides in length. 84. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 260 nucleotides in length. 85. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 270 nucleotides in length. 86. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 280 nucleotides in length. 87. The method or cell, according to any of the previous embodiments, wherein said GRON is 290 nucleotides in length. 88. The method or cell, according to any of the previous embodiments, wherein said GRON is 300 nucleotides in length. 89. The method or cell, according to any of the previous embodiments, wherein said GRON is 400 nucleotides in length. 90. The method or cell, according to any of the previous embodiments, wherein said GRON is 500 nucleotides in length. 91. The method or cell, according to any of the previous embodiments, wherein said GRON is 600 nucleotides in length. 92. The method or cell, according to any of the previous embodiments, wherein said GRON is 700 nucleotides in length. 93. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 800 nucleotides in length. 94. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 900 nucleotides in length. 95. The method or cell, according to any of the previous embodiments, wherein said GRON is more than 1000 nucleotides in length. 96. The method or cell, according to any of the preceding embodiments, in which said plant is selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, corn, wheat, barley, rice , alfalfa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, soybean, soybean spp, sugar cane, pea, chickpea, pea, broad beans, lentils, turnips, rutabaga, Brussels sprouts, lupins, cauliflower, cabbage, broad beans, poplar, pine, eucalyptus, grapes, citrus, triticale, alfalfa, rye, oats, grass and forage grasses, flax, rapeseed, mustard , cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, cassava and lily. 97. The method or cell, according to any of the previous embodiments, wherein said plant is canola. 98. The method or cell, according to any of the previous embodiments, wherein said plant is corn. 99. The method or cell, according to any of the previous embodiments, wherein said plant is corn. 100. The method or cell, according to any of the previous embodiments, wherein said plant is rice. 101. The method or cell, according to any of the previous embodiments, wherein said plant is sorghum. 102. The method or cell, according to any of the previous embodiments, wherein said plant is potato. 103. The method or cell, according to any of the previous embodiments, wherein said plant is soybeans. 104. The method or cell, according to any of the previous embodiments, wherein said plant is flax. 105. The method or cell, according to any of the previous embodiments, wherein said plant is rapeseed. 106. The method or cell, according to any of the previous embodiments, wherein said plant is cassava. 107. The method or cell, according to any of the previous embodiments, wherein said plant is sunflower. 108. Method for causing a genetic change in a plant cell, said method comprising exposing said cell to a CRISPRS and a modified GRON. 109. The method or cell, according to any of the previous embodiments, in which multiple genetic changes are carried out. 110. The method or cell, according to any of the previous embodiments, in which two or more guide RNAs are used. 111. The method or cell, according to any of the preceding embodiments, in which each of the more than one guide RNA is complementary to a different target for genetic alteration. 112. The method or cell, according to any of the preceding embodiments, in which CRISPRS includes a nickase. 113. The method or cell, according to any of the preceding embodiments, wherein the DNA cutter includes two or more nickases. 114. The method or cell, according to any of the preceding embodiments, in which two or more nickases cleave on opposite strands of the target nucleic acid sequence. 115. The method or cell, according to any of the preceding embodiments, in which two or more nickases cleave on the same strand of the target nucleic acid sequence. 116. A non-transgenic herbicide resistant or tolerant plant made by the method or from the cell of any of the preceding embodiments. 117. The method or cell of any of the preceding embodiments, wherein said plant cell has a genetic change or mutation in acetyl coenzyme A carboxylase (ACCase) and is selected from the group consisting of barley, corn, millet, oats , rye, rice, sorghum, sugar cane, grasses and wheat. 118. The method or cell of any of the preceding embodiments, wherein said plant cell has a genetic change or mutation in acetyl coenzyme A carboxylase (ACCase) and is resistant or tolerant to one or more herbicides. 119. The method or cell of any of the preceding embodiments, wherein said plant cell has a genetic change or mutation in acetyl coenzyme A carboxylase (ACCase), is resistant to one or more herbicides that inhibit ACCase. 120. The method or cell of any of the preceding embodiments, wherein said plant cell has a genetic change or mutation in acetyl coenzyme A carboxylase (ACCase), is resistant to one or more herbicides selected from the group consisting In alxidim, buttxydim, cletodim, cloproxidim, cycloxidy, setoxidim, tepraloxidy, tralcoxidima, clodinafop, clodinafop, diclofop, fenoxrop-p, fentiaprop, fluazifop, fluazifop-P, haloxifop, haloxifop, isoxapp FOP, Propaquizafop, Quizalofop , quizalofop-P, trifop, pinoxaden, agronomically acceptable salts and esters of any of these herbicides, and combinations thereof. 121. The method or cell of any of the preceding embodiments, wherein said plant cell has a genetic change or mutation in 5-enolpyruvylxykimate-3-phosphate synthase (EPSPS), and wherein said plant cell is selected from the group which consists of corn, wheat, rice, barley, sorghum, oats, rye, sugar cane, soybeans, cotton, sugar beet, rapeseed, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus , apple, lettuce, peas, lentils, grapes and grasses. 122. The method or cell of any of the preceding embodiments, wherein said plant cell or plant has a genetic change or mutation in 5-enolpyruvylxykimate-3-phosphate synthase (EPSPS), and wherein the plant cell or plant is resistant to at least one herbicide. 123. The method or cell of any of the preceding embodiments, wherein said plant cell or plant has a genetic change or mutation in 5-enolpyruvylxykimate-3-phosphate synthase (EPSPS), and wherein the plant or plant cell is resistant to a herbicide from the phosphonomethylglycine family. 124. The method or cell of any of the preceding embodiments, wherein said plant cell or plant has a mutation or genetic change in 5-enolpyruvylxykimate-3-phosphate synthase (EPSPS), and wherein the plant cell or plant is glyphosate resistant. 125. The method or cell of any of the preceding embodiments, wherein said plant or plant cell has a genetic change or mutation in 5-enolpyruvylxykimate-3-phosphate synthase (EPSPS), and wherein the plant or plant cell is selected from the group consisting of corn, wheat, rice, barley, sorghum, oats, rye, sugar cane, soybeans, cotton, sugar beet, rapeseed, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa , poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grapes and grasses. 126. The method or cell of any of the above embodiments, in which the genetic change or mutation in the cell occurs in one allele of the gene. 127. The method or cell of any of the above embodiments, in which the genetic change or mutation in the cell occurs in two alleles of the gene. 128. The method or cell of any of the above embodiments, in which the genetic change or mutation in the cell occurs in three alleles of the gene. 129. The method or cell of any of the above embodiments, in which the genetic change or mutation in the cell occurs in four alleles of the gene. 130. The method or cell of any of the preceding embodiments, in which the genetic change or mutation in the cell occurs in one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve alleles of the gene. 131. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell comprises a deletion or insertion, resulting in a knockout of an allele of the gene. 132. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell comprises a deletion or insertion, resulting in a knockout of two alleles of the gene. 133. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell comprises a deletion or insertion, resulting in a knockout of three alleles of the gene. 134. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell comprises a deletion or insertion, resulting in a knockout of four alleles of the gene. 135. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of one, two, three, four, five, six, seven, eight, nine , ten, eleven, or twelve alleles of the gene. 136. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell occurs in one allele of the gene and a second allele of the gene comprises a deletion or insertion, resulting in a knockout of said second allele. 137. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell occurs in one allele of the gene and a second allele and third allele of the gene comprises a deletion or insertion, resulting in a knockout of said second allele and said third allele. 138. The method or cell of any of the preceding embodiments, wherein the genetic change or mutation in the cell occurs in one allele of the gene and a second allele, third allele and fourth allele of the gene comprises a deletion or insertion, resulting in a knockout of said second allele, said third allele and said fourth allele. 139. The method or cell of any of the preceding embodiments, wherein the genetic change in the cell comprises at least one mutation in one allele and at least knockout in another allele. 140. The method or cell of any of the preceding embodiments, wherein the genetic change in the cell comprises at least one mutation in one allele and at least one knockout in at least one other allele. 141. The method or cell of any of the preceding embodiments, wherein the genetic change in the cell comprises at least one mutation in one allele and at least knockout in at least two other alleles. 142. The method or cell of any of the preceding embodiments, wherein the genetic change in the cell comprises at least one mutation in one allele and at least knockout in at least three other alleles. 143. The method or cell of any of the preceding embodiments, wherein the genetic change in the cell comprises at least one mutation in one allele and knockout in all other alleles.

EXAMPLES

[0273] The following are examples, which illustrate procedures for practicing the methods and compositions described herein. These examples should not be construed as limiting.

[0274] Example 1: GRON length

[0275] Sommer et al., (Mol Biotechnol. 33: 115-22, 2006) describes a reporter system for the detection of gene conversion in vivo that relies on 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 evaluate GRON conversion efficiency following GRON length modification.

[0276] In summary, for this and subsequent examples an Arabidopsis thaliana line with multiple copies of a blue fluorescent protein gene was created by methods known to those skilled in the art (see, for example, Clough and Brent, 1998). Root-derived meristematic tissue cultures were established with this line, in which it was used for the isolation and culture of protoplasts (see, e.g., Mathur et al., 1995). The distribution of GRON into protoplasts was achieved through polyethylene glycol (PEG) upon admission of GRON into protoplasts. A method using a 96-well format, similar to that described by Kato and Fujiwara (2007), was used. Next, the protocol is briefly described. The volumes indicated are those applied to individual wells of a 96-well plate. 1. Mix 6.25 μl of GRON (80 μM) with 25 μl of meristematic tissue-derived protoplasts from the BFP transgenic root of Arabidopsis thaliana at 5x106 cells/ml in each well of a 96-well plate. 2. 31.25 μl 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 μl of W5 solution was added and the cells mixed. 5. Plates were allowed to incubate on ice for 30 min, allowing protoplasts to settle to the bottom of each well. 6. 200 μl of the medium above the established protoplasts was removed. 7. 85 μl of culture medium (MSAP, see Moraes et al., 1995) was added. 8. The plates were incubated at room temperature in the dark for 48 hours. The final concentration of GRON after addition of the culture medium is 8 μM.

[0277] Forty-eight hours after Gron distribution samples were analyzed by flow cytometry in order to detect protoplasts whose green and yellow fluorescence is different from those of control protoplasts (BFP0 indicates non-bleaching GRONs with no change compared to BFP target; C is the coding tape design and NC is the non-coding tape design). A single C to T nucleotide difference (coding strand) or G to A nucleotide target mutation (non-coding strand) in the center of BFP4 molecules. Green fluorescence is caused by the introduction of a target mutation in the BFP gene, resulting in the synthesis of GFP. The results are shown in Figure 1.

[0278] Table 2 shows example sequences 101-mer and 201-mer BFP4/NC 5'-3PS/ 3'-3PS GRONs designed for converting a blue fluorescent protein (BFP) gene to green fluorescence. "3PS" indicates 3 phosphothioate bonds at each of the 5' and 3' oligo ends.

[0279] Table 2: Examples of GRON Nucleotide Sequences for converting BFP to GFP * = PS (phosphorothioate) binding

[0280] Example 2: Conversion rates using GRONs marked with 5’Cy3/ 3’idC

[0281] The objective of this series of examples is to compare the efficiency of GRONs labeled with phosphorothioate (PS) (having fractions of 3 PS at each end of GRON) to GRONs labeled with 5'Cy3/3'idC. 5'Cy3/3'idC-labeled GRONs have a 5' Cy3 (amidite) fluorophore and a 3' idC reverse base. Efficiency was assessed using the conversion of blue fluorescent protein (BFP) to green fluorescence.

[0282] In all three examples, made either by distributing PEG from GRONs into the protoplasts in individual Falcon tubes (labeled "Tubes") or in 96-well plates (labeled "96-well dish"), there was no significant difference between the different GRON chemistries on the conversion efficiency of BFP to GFP, as determined by cytometry (Figure 1).

[0283] Example 3: Comparison between GRON BFP4/NC 5'- 3PS/ 3'-3PS and 2'-O-Me 41-mer GRONs

[0284] The purpose of this series of examples is to compare the conversion efficiencies of phosphorothioate (PS)-labeled GRONs with 3PS moieties at each end of GRON to 2'-O-Me or "Okazaki fragment GRONs" in the presence and absence of a member of the bleomycin family, Zeocin™ (1 mg/ml) to induce DNA breaks. The designs of these GRONs are represented in Figure 2. The GRONs were delivered into Arabidopsis thaliana BFP protoplasts upon PEG treatment and conversion of BFP to GFP was determined 24h after treatment by cytometry. Samples treated with zeocin (1 mg/ml) were incubated with zeocin for 90 min on ice before PEG treatment.

[0285] In general, the presence of zeocin (1 mg/ml) increased the conversion of BFP to GFP, as determined by cytometry (Table 3). Both in the presence and absence of zeocin, NC Okazaki's GRON containing a 2'-O Me group in the first RNA base at the 5' end of the GRON was more effective in converting BFP to GFP when compared to NC Okazaki's GRON. containing a 2'-O Me group in each of the first nine 5' RNA bases (Figure 2 and Table 3).

[0286] In all examples, there was no significant difference between the 41-mer BFP4/NC 5'3PS/ 3'3PS and the GRON of BFP4/NC from the 71-mer Okazaki Fragment that contains a 5' 2'- group. The me at the 5' first RNA base (denoted as BFP4 71-mer(1)NC) in conversion of BFP to GFP in either the presence or absence of 1 mg/ml zeocin as determined by cytometry (Figure 2 and Table 3). It is important to note that in the presence of zeocin (and expected for bleomycin, phleomycin, tallisomycin, pepleomycin and other members of this family of antibiotics) that the conversion becomes strand independent (i.e., both GRONs encoding (C) and not coding (NC) with the models tested in these examples exhibit approximately equal activity).

[0287] Table 3: Comparison of a standard GRON design with the Okazaki fragment GRON design fragment in the presence and absence of a zeocin glycopeptide antibiotic.

[0288] Example 4: Comparison between GRONs 41-mer, 101-mer and 201-mer BFP4/NC 5'-3PS/3'-3Ps

[0289] The purpose of this series of examples was to compare the conversion efficiencies (in the presence and absence of zeocin) of phosphorothioate (PS)-labeled GRONs with 3PS moieties at each end of the GRON of different lengths: 41-mer, 101 -mer and 201-mer shown in Table 2. Again, the presence of zeocin (1 mg/ml) increased the conversion rate of BFP to GFP, as determined by cytometry (Table 4). The general trend in all three examples was linear with increasing GRON NC length in both the presence and absence of zeocin. Except for BFP-4/NC/101 and BFP-4/C/101, in the presence of zeocin, this had conversion rates that were close to equal to, but lower than, GRON 41-mer NC. This is in contrast to all previous examples in which BFP-4/41 coding and non-coding GRONs were used, where the non-coding GRON was always much superior to the coding one. This asymmetry in conversion frequency also applies to the BFP-4/201 GRONs used in this series of examples.

[0290] Table 4:

[0291] Example 5: Distribution of Cas9 Protein in Plants

[0292] This example makes use of direct delivery of recombinant Cas9 protein to plant cells as an alternative to the delivery of CRISPRS-Cas expression plasmids. This method employs carriers such as cell penetrating peptides (CPP), liposome transfection reagents, poly(ethylene glycol) (PEG) either alone or in combination to enable delivery of active recombinant Cas9 protein to cells.

[0293] Methods

[0294] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. Fluorescently labeled recombinant Cas9 protein (1 μg) pre-coated with CPPs at 20:1, 10: or 5:1 and another CPP to charge ratio (TAT, Penetratin, Chariot™, PEP-1 or others, e.g. ) or encapsulated with liposome reagents are then mixed with the seeded protoplasts and incubated at 23°C for 2-6 hours to allow the Cas9 complexes/transporters to penetrate the cells. In another series of treatments fluorescently labeled recombinant Cas9 protein (1 μg) either pre-coated with CPPS as described above or uncoated are introduced to protoplasts using PEG methodology. The protoplasts were then analyzed by flow cytometry 24h after treatment in order to determine the percentage of Cas9 positive protoplasts within a given treatment.

[0295] Example 6: CRISPR with 201-mer GRONs ± oscillation basis

[0296] The purpose of this series of examples is to demonstrate conversion of BFP to GFP in our Arabidopsis thaliana BFP transgenic model system using CRISPRs to create target double-strand breaks in the bfp gene and the 201-mer GRONs to mediate the conversion. CRISPRS BFP targets the coding strand of the bfp gene and the conversion site is 27 bp upstream of the PAM sequence (Figure 3). GRON is used as a template to repair the double-strand break in the bfp gene created by CRISPRS and together with the conversion of the target gene from BFP to GFP, a wobble base is introduced into the bfp gene that corresponds to the PAM sequence of CRISPR BFP too. One basis for oscillation in the CRISPR BFP PAM sequence is the hypothesis to minimize re-cutting of the bfp gene by CRISPRs once conversion has occurred. This series of examples will help address whether or not introducing a wobble base into the CRISPR BFP PAM sequence in converted bfp genes will increase conversion efficiency.

[0297] Methods

[0298] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue were seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-encoded plasmids contain the mannopine synthase (MAS) promoter driving the Cas9 coding sequence with an rbcSE9 terminator and the Arabidopsis U6 promoter driving the sgRNA with a poly-T10 terminator. CRISPRS plasmids together with GRONs were introduced into protoplasts upon PEG-mediated delivery at a final concentration of 0.05 μg/μl and 0.16 μM respectively. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0299] CRISPR consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. In this example, BFP targets the bfp gene (Figure 3). 201-mer GRONs that target BFP with or without the wobble bases were used to determine their effect on the conversion rate of BFP to GFP. Table 5 provides a list of GRONs and their corresponding sequences.

[0300] Table 5. List of GRONs used in these examples

[0301] Results:

[0302] Using CRISPR BFP, GRON BFP4/C with the wobble bases is up to 3.5 times more effective at converting BFP to GFP when compared to GRON BFP4/C without the wobble bases (Table 6). There is a 5.9-fold increase in the conversion of BFP to GFP when GRON BFP4/C with the wobble base is used instead of GRON BFP4/CN with the wobble base (Table 6). Therefore, GRON BFP4/C with the oscillation base is more effective in converting BFP to GFP when used with CRISPR BFP.

[0303] Conclusions

[0304] Including a wobble base in GRON that alters the CRISPRS BFP PAM sequence in the converted target gene increases conversion of BFP to GFP. The conversion of BFP to GFP by CRISPRS BFP together with GRON oscillation base was confirmed by Next Generation Sequencing (data not shown). Furthermore, the ability of CRISPRS BFP to cleave DNA and produce the bfp gene was confirmed by Next Generation Sequencing (data not shown).

[0305] Table 6. The percentage of conversion of BFP to GFP as determined by cytometry at 72 hours post PEG distribution of CRISPR BFP and GRON in protoplasts derived from BFP transgenic line Arabidopsis thaliana 21-15-09.

[0306] Example 7: CRISPR with Cy3-modified GRONs

[0307] The purpose of this series of examples is to demonstrate conversion of BFP to GFP in our BFP Arabidopsis thaliana transgenic model system using CRISPRS to create targeted double-strand breaks in the bfp gene and the GRONs to mediate the conversion. The CRISPR BFP6 (CR:BFP6) used in these examples targets the bfp gene and causes a double-strand break in the DNA near the conversion site. The GRONs used with CRISPRS BFP6 contain the bfp gene coding sequence around the conversion site and are labeled at the 5' end with Cy3 and at the 3' end with an idC reverse base and are referred to here as Cy3 GRONs. These GRONs are tested in three different lengths of 41-mers, 101-mers and 201-mers and are directly compared to 3PS GRONs which only differ from Cy3 GRONs in that they have 3 phosphothioate bonds at both the 5' and 3' ends. from GRON. These GRONs are referred to here as 3PS GRONs. See Table 7 for the list of GRONs used in these examples

[0308] Methods

[0309] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue were seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-encoded plasmids contain the MAS promoter that targets the Cas9 coding sequence with an rbcSE9 terminator and Arabidopsis thaliana U6 promoter that targets the sgRNA with a poly-T10 terminator. CRISPRS plasmids together with GRON were introduced into protoplasts via PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 8.0 μM for 41-mer GRONs, 0.32 μM for 101-mer GRONs. mer and 0.16 μM 201-mer. GRON treatments alone received a final GRON concentration after PEG delivery of 8.0 μM for the 41-mer, 5.0 μM for the 101-mer, and 2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0310] CRISPRS consists of two components: Cas9 (SpCas9) and sgRNA from Streptococcus pyogenes with optimized plant codon, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. In this experiment, the CRISPRS BFP6 (5'GGTGCCGCACGTCACGAAGTCGG 3') was used, which targets the bfp gene. GRONs contain the bfp gene coding sequence near the conversion site. Table 7 presents a list of the GRONs used.

[0311] Table 7. List of GRONs used in these examples

[0312] Results:

[0313] Using CRISPR BFP6, Cy3 GRONs at all lengths tested were able to mediate BFP to GFP conversion as efficiently as 3PS GRONs (Figure 4). In general, samples containing the CRISPR BFP6 and GRON have higher levels of BFP to GFP conversion when compared to GRON-only samples (Figure 4), demonstrating the positive impact of CRISPRS on increasing conversion rates.

[0314] Example 8: CRISPRS with variable-sized GRONs

[0315] The purpose of this series of examples is to demonstrate conversion of BFP to GFP in our BFP Arabidopsis thaliana transgenic model system using CRISPRs to create target double-strand breaks in the bfp gene and GRONs of variable lengths to mediate the conversion. The CRISPRS BFP used in this example targets the bfp gene and causes a double-strand break in the DNA near the conversion site. The GRONs used with CRISPR BFP, contain the bfp gene coding sequence around the conversion site and are labeled at both the 5' end and the 3' end with 3 phosphothioate bonds and are referred to here as 3PS GRONs. These GRONs are tested in three different lengths of 60-mers, 101-mers and 201-mers and are directly compared to GRON-only treatments. See Table 8 for the list of GRONs used in these examples.

[0316] Methods

[0317] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue were seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-encoded plasmids contain the MAS promoter driving the Cas9 coding sequence with an rbcSE9 terminator and the Arabidopsis thaliana U6 promoter driving the sgRNA with a poly-T10 terminator. CRISPRS plasmids together with GRON were introduced into protoplasts via PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 0.547 μM for 60-mer GRONs, 0.32 μM for 101-mer and of 0.16 μM 201-mer. GRON treatments alone received a final GRON concentration after PEG delivery of 7.5 μM for the 60-mer, 5.0 μM for the 101-mer, and 2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0318] CRISPR consists of two components: Cas9 (SpCas9) and sgRNA from Streptococcus pyogenes with optimized plant codon, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. The CRISPR BFP sequence is 5’GTCGTGCTGCTTCATGTGGT3’. In this example, CRISPR BFP was used which targets the bfp gene. GRONs contain the bfp gene coding sequence near the conversion site. Table 8 presents a list of the GRONs used.

[0319] Table 8. List of GRONs used in these examples.

[0320] Results:

[0321] Using CRISPR BFP, GRONs in lengths >101 nt are better at mediating conversion of BFP to GFP when compared directly to GRONs of 60 nt in length (Figure 5). In general, samples containing the CRISPR BFP and GRON have higher levels of BFP to GFP conversion when compared to GRON-only samples (Figure 5), demonstrating the positive impact of CRISPRS on increasing conversion rates. These data further demonstrate that the length of GRON that is most effective in mediating BFP to GFP conversion, when used in conjunction with CRISPR, needs to be >101 nt in length.

[0322] Example 9: CRISPR with 2’-O-Me GRONs

[0323] The purpose of this example is to demonstrate the conversion of BFP to GFP in our BFP Arabidopsis thaliana transgenic model system using CRISPRs to create target double-strand breaks in the bfp gene and GRONs to mediate the conversion. The CRISPR BFP used in this example targets the bfp gene and causes a double-strand break in the DNA near the conversion site. The GRONs used with CRISPR BFP contain either the coding or non-coding sequence of the bfp gene around the conversion site with the first ten bases in the 5' of GRON being RNA bases rather than DNA bases. These RNA bases are labeled with 2'-O-Me group(s) either the 5' first RNA base or the 5' first RNA base as depicted in Figure 6. These GRONs are referred to here as GRONs 2 '-O-Me and are directly compared to 3PS GRONs of similar lengths that contain DNA bases with 3 phosphothioate linkages at both the 5' and 3' ends of GRON. These GRONs are referred to here as 3PS GRONs. See Table 9 for the list of GRONs used in these examples

[0324] Methods

[0325] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue were seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. The encoded CRISPR plasmids contain the MAS promoter, which drives the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis U6 promoter, which drives the sgRNA with a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). CRISPR plasmids together with GRON were introduced into protoplasts via PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPR, 0.5 μM for 71-mer GRONs, and 0.16 μM for 201-mer GRONs. . GRON treatments alone received a final GRON concentration after PEG delivery of 5.0 μM for the 71-mer and 2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0326] CRISPR consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. The CRISPRS BFP spacer sequence is 5'CTCGTGACCACCTTCACCCA 3'. In this example, the CRISPRS BFP was used which targets the bfp gene. GRONs contain any coding or non-coding sequence of the BFP gene near the conversion site. Table 9 shows a list of the GRONs used.

[0327] Table 9. List of GRONs used in these examples

[0328] Results:

[0329] The 2'-O-Me 71-mer and 201-mer GRONs had similar BFP to GFP conversion when compared to the various different types of GRON protections of (0), (1) or (9) using CRISPR BFP (Figures 7 and 8). The 2'-O-Me GRONs are more effective than their GRON 3PS counterparts in mediating the conversion of BFP to GFP using the BFP CRISPRSs (Figures 7 and 8).

[0330] Example 10: CRISPRS Nickases with Introduction of GRONs

[0331] The purpose of this example is to demonstrate the conversion of BFP to GFP in our BFP Arabidopsis thaliana transgenic model system using CRISPRs to create target single-strand breaks in the bfp gene and GRONs to mediate the conversion. The CRISPRS BFP1 (CR:BFP1) used in this example targets the bfp gene and contains mutations in the catalytic residues (D10A in RuvC and H840A in HNH) that cause single-strand cuts in the bfp gene DNA near the conversion site or at the DNA from complementary or non-complementary strands to the guide RNA, respectively. These CRISPRS are referred to here as D10A nickase CRISPR BFP1 and H840A nickase CRISPRS BFP1 and are used alone or with BFP5 sgRNA on a separate plasmid. When multiple CRISPR nickases are used together in this example, they can either cut the same strands of DNA or strands of opposite DNA. When both Cas9 proteins that contain the same mutations, either D10A or H840A, are used together, they cut the same strand of DNA. On the other hand, when two Cas9 proteins are used together and one of them contains the D10A mutation and the other contains the H840A mutation, they cut opposite strands of DNA. The GRONs used with CRISPRS nickase contain either the coding or non-coding sequence of the bfp gene around the conversion site with a wobble base located in the CRISPRS BFP5 PAM sequence. These GRONs have 3 phosphothioate linkages at both the 5' and 3' ends and are referred to here as 3PS GRONs. See Table 10 for the list of GRONs used in these examples. The CRISPR nickases are directly in comparison to their CRISPR counterparts that are capable of causing targeted double-strand breaks in the DNA of the bfp gene.

[0332] Methods

[0333] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-encoded plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis thaliana U6 promoter, driving the sgRNA with a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 gene contains mutations in the catalytic residues, either D10A in RuvC or H840A in HNH. CRISPRS plasmids along with GRON are introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 0.16 μM for the 201-mer GRONs. GRON treatments alone received a final GRON concentration after PEG delivery of 2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0334] CRISPR consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. In this example, the BFP1 and BFP5 sgRNA was used that targets different regions of the bfp gene near the conversion site. The BFP1 spacer (5'CTCGTGACCACCTTCACCCA 3') targets the coding strand while the BFP5 spacer (5'GTCGTGCTGCTTCATGTGGT3') targets the non-coding strand of the bfp gene. GRONs contain any coding or non-coding sequence of the BFP gene near the conversion site. Table 10 shows a list of the GRONs used.

[0335] Table 10. List of GRONs used in these examples

[0336] Results:

[0337] Both CRISPR nickases (D10A and H840A) are more efficient in mediating BFP to GFP conversion when CRISPRS BFP1 and CRISPRS BFP5 were used together rather than separately (Figure 9). Furthermore, when the D10A CRISPRS nickases BFP1 and BFP5 are used together with the GRON 1W C/201 ratio, the BFP to GFP conversion is significantly higher when compared to treatments where these CRISPRS nickases are used with the GRON 1W NC/ 201 (Figure 9). When the CRISPRS H840A nickases BFP1 and BFP5 are used together more or less the same degree of BFP to GFP conversion is observed with either 1W C/201 or NC/201 GRONs (Figure 9). These levels of BFP to GFP conversion are slightly higher than when CRISPRS BFP5 is used alone and slightly lower than when CRISPRS BFP1 is used alone (Figure 9).

[0338] Example 11: Use of CRISPRs to Target Multiple Genes

[0339] The objective of this example is to demonstrate the conversion of multiple genes simultaneously in a given population of protoplasts derived from the Arabidopsis thaliana model system using CRISPRs to create double-strand breaks in target genes and GRONs to mediate conversion. The CRISPRS used in this example target both the BFP and acetohydroxy acid synthase genes (AHAs) in the Arabidopsis thaliana genome by introducing into protoplast plasmids encoding the Cas9 gene and multiple sgRNAs that target these two different genes. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). This will allow Cas9 to cause double-strand breaks in both BFP and AHAS genes in the presence of GRONs that will mediate their conversion.

[0340] Methods

[0341] Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. The CRISPRS-encoded plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis thaliana U6 promoter, driving multiple different sgRNAs with a poly-T10 terminator. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). CRISPRS plasmids along with GRON are introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 0.16 μM for the 201-mer GRONs. GRON treatments alone receive a final GRON concentration after PEG delivery of 2.5 μM to the 201-mer. Protoplasts will be incubated in the dark at 23°C for 72 hours and then analyzed by flow cytometry and an allele-specific PCR assay in order to determine the percentage of both protoplasts converted from BFP to GFP and from AHAS. , respectively, within a given treatment.

[0342] In an allele-specific PCR assay 10-16 replicates of 5,000 genome equivalents of genomic DNA were used in the primary PCR reactions.

[0343] CRISPRS consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same or multiple plasmids. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to target genes. In this example different sgRNAs and GRONs are used to target multiple genes near their conversion sites; BFP spacer (5’CTCGTGACCACCTTCACCCA 3’) and AHAS spacer (5’ TGGTTATGCAATTGGAAGATCGC 3’). Table 11 describes the GRONs used.

[0344] Table 11. List of GRONs used in this example

[0345] Results:

[0346] The conversion of BFP to GFP and AHAS was determined 144h after PEG distribution of the CRISPRS plasmids BFP and AHAS and GRONs BFP/C 201-mer and AHAS(W)574/NC 201-mer in a BFP transgenic line Arabidopsis thaliana. Flow cytometry data revealed that Treatment 1 resulted in 0.20% conversion of BFP to GFP (Table 12). The allele-specific PCR assay revealed that Treatment 1 resulted in 0.01% protoplasts converted from AHAS (Table 12). GRON-only treatments had minimal conversion using both assays (Table 12). This example demonstrates the successful simultaneous conversion of two independent target genes (BFP and AHAS) within a given population of protoplasts derived from the Arabidopsis thaliana BFP model system.

[0347] Table 12. Measurement of gene conversion of both BFP and AHAS at 144h after PEG delivery of CRISPRS and GRONs plasmids in a given population of protoplasts derived from the BFP Arabidopsis thaliana model system or by: (1) flow cytometry which determines the percentage of GFP-positive protoplasts or (2) allele-specific PCR which determines the percentage of AHAS-converted protoplasts.

[0348] Example 12: Delivery of Cas9 mRNA into Plant Cells

[0349] This example makes use of direct delivery of recombinant Cas9 mRNA to plant cells as an alternative to the delivery of CRISPRS-Cas expression plasmids. This method includes (1) in vitro synthesis of modified mRNA and (2) Delivery of this modified mRNA into plant cells.

[0350] Methods

[0351] A Cas9 mRNA will be transcribed in vitro using an RNA polymerase, such as T7, T3 or SP6 from a linearized plasmid template including components of a 5'UTR, the coding sequence (CDS) for the protein and a 3' UTR. An RNA polymerase can incorporate a particular modified nucleoside better than another. 5' UTR may contain elements that improve its stability, such as MiR-122 of the hepatitis C virus (Shimakami et al., 2012). In vitro synthesis will incorporate nucleosides that are protective and ensure good translation into target plant cells. Recombinant Cas9 mRNA will be capped and contain a polyA tail.

[0352] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. Recombinant Cas9 mRNA will be delivered to plant cells in one of the following media (list not inclusive): cell penetrating peptides (CPP), liposome transfection reagents, poly(ethylene glycol) (PEG), or alone or in combination to allow delivery of active recombinant Cas9 mRNA into cells.

[0353] Example 13: CRISPRS-Cas to trap DNA, RNA or proteins.

[0354] This example makes use of a modified single guide RNA (sgRNA) cassette, in which a linker sequence (may also be referred to as a tethering sequence) is included at the 3' end of tracrRNA but 5' of the tracrRNA signal. termination of RNA polymerase III, as shown in the example below (Figure 10). The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). Although preferred, linker positioning is not limited to the 3' end of tracerRNA, but will be investigated at various positions within the sgRNA cassette. The linker sequence may vary in nucleotide length or contain a secondary structure that could better tether or increase the number of tethered molecules through triplex interactions.

[0355] The linker will allow Watson-Crick base pairing with DNA, RNA or proteins that contain the complementary sequence (Figure 10). Furthermore, the sequence linker in sgRNA cassettes will be designed to contain advanced secondary and tertiary structure allowing for more complex multifaceted interaction regions that would tether multiple DNA, RNA, or protein molecules.

[0356] The general concept is that a CRISPRS-Cas complex will tether biological molecules to the site of nuclease activity, thereby increasing the likelihood of gene editing. These biological molecules include GRON which will mediate the conversion of target gene(s). Tethering linkers can be added to the sgRNA simply using, for example, Gene Strings or the hybridized oligos.

[0357] Methods

[0358] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-Cas anchoring plasmids contain the MAS promoter that drives the Cas9 coding sequence with an rbcSE9 terminator and

[0359] Arabidopsis thaliana U6 promoter that drives sgRNAs with a linker sequence that is complementary to a 15-30 bp polynucleotide tract located in a 201-mer that edits GRON that targets bfp (as shown in Figure 10). The sgRNA tethering cassette is terminated by a poly-T10 terminator. CRISPRS-Cas plasmids along with GRON are introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 0.16 μM for the 201-mer GRON. GRON treatments alone received a final GRON concentration after PEG delivery of 2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23°C for 72 hours and were then analyzed by flow cytometer in order to determine the percentage of GFP positive protoplasts within a given treatment.

[0360] CRISPR consists of two components: Cas9 (SpCas9) and sgRNA from Streptococcus pyogenes with optimized plant codon, both of which are expressed from the same or different plasmids. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) and linker. The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. In this example, CRISPRS targeting the bfp gene was used. GRONs contain any coding or non-coding sequence of the bfp gene near the conversion site.

[0361] Example 14: CRISPRs with truncated gRNA.

[0362] The purpose of this example is to demonstrate the conversion of BFP to GFP in protoplasts derived from the Arabidopsis thaliana BFP model system using CRISPRs to create double-strand breaks in target genes and GRONs to mediate conversion. The CRISPRs used in this example target the bfp gene in the Arabidopsis thaliana genome by introducing into protoplast plasmids that encode the Cas9 gene and sgRNAs that are two different lengths. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA that guides Cas9 to target genes is called a spacer and is typically 20-nt in length (CR:BFP1 20-nt), however, in these examples the effectiveness of using a spacer of shorter length than 17 was tested. - nt (CR:BFP1 17-nt) in mediating the conversion of BFP to GFP.

[0363] Methods

[0364] Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. The CRISPRS-encoded plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis thaliana U6 promoter, driving multiple different sgRNAs with a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). CRISPR plasmids along with GRON are introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPR and 0.16 μM for the 201-mer GRONs. GRON treatments alone receive a final GRON concentration after PEG delivery of 2.5 μM to the 201-mer. The protoplasts will be incubated in the dark at 23 °C for 72 hours and then they will be analyzed by flow cytometer in order to determine the percentage of BFP to GFP within a given treatment.

[0365] CRISPRS consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same or multiple plasmids. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to target genes. In these examples, two BFP1 spacers of different lengths of 20-nt (5'CTCGTGACCACCTTCACCCA 3') vs. 17-nt (5'GTGACCACCTTCACCCA 3') were tested. Table 13 describes the GRON used

[0366] Table 13. List of GRON used in this example

[0367] Results:

[0368] Reducing the length of the BFP1 protospacer from 20 bp to 17 bp had similar levels of BFP to GFP conversion of 0.163% vs 0.177%, respectively, at 72h after PEG delivery of the plasmids and GRONs in the Arabidopsis thaliana BFP model system (Figure 11).

[0369] Example 15: CRISPRs with gRNA amplicon.

[0370] The purpose of this example is to demonstrate the conversion of BFP to GFP in protoplasts derived from the Arabidopsis thaliana BFP model system using CRISPRs to create double-strand breaks in target genes and GRONs to mediate conversion. The CRISPRs used in this example target the bfp gene in the Arabidopsis thaliana genome through introduction into plasmids of protoplasts encoding the Cas9 gene and sgRNAs that are either encoded on a plasmid or introduced into protoplasts with an amplicon. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA guides Cas9 to target genes, where Cas9 creates a double-strand break and GRON is used as a template to convert from BFP to GFP in a site-directed manner.

[0371] Methods

[0372] Arabidopsis thaliana protoplasts derived from induced root tissue are seeded in a flat-bottom 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPR-encoded plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis U6 promoter, driving multiple different sgRNAs with a poly-T10 terminator. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). CRISPRS plasmids along with GRON are introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μl for CRISPRS and 0.16 μM for the 201-mer GRONs. GRON treatments alone receive a final GRON concentration after PEG delivery of 2.5 μM to the 201-mer. The protoplasts will be incubated in the dark at 23 °C for 72 hours and then they will be analyzed by flow cytometer in order to determine the percentage of BFP to GFP within a given treatment.

[0373] CRISPR consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from the same or multiple plasmids. The sgRNA is a fusion of CRISPRS RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to target genes. In these examples, the same BFP6 gRNA (5'GGTGCCGCACGTCACGAAGTCGG 3') was delivered into protoplasts both as an amplified fragment and encoded on a plasmid. Table 14 describes the GRONs used.

[0374] Table 14. List of GRONs used in this example

[0375] Results

[0376] Delivery of the BFP6 gRNA as an amplified fragment (CR: BFP6 (gRNA amplicon)) together with a plasmid containing only Cas9 had similar rates of BFP to GFP conversion when compared to treatments of both gRNA (gRNA plasmid) and Cas9 be encoded on separate plasmids at 72h after PEG plasmid delivery and for GRONs in the exemplary Arabidopsis thaliana BFP system (Figure 12).

[0377] Example 16: CRISPRs with unmodified GRONs.

[0378] The purpose of this example is to demonstrate BFP to GFP conversion in our Arabidopsis thaliana BFP transgenic model system using CRISPR to create targeted double-strand breaks in the bfp gene and GRONs to mediate conversion. The CRISPRS BFP used in this example targets the bfp gene and causes a double-strand break in the DNA near the conversion site. 3PS GRONs contain DNA bases with 3 phosphothioate linkages at both the 5' and 3' ends of the GRON and are referred to here as 3PS GRONs. The 3Ps GRONs were directly compared with their unmodified Gron counterparts in mediated conversion of BFP to GFP using the BFP CRISPRS in our Arabidopsis thaliana BFP transgenic model system. See Table 15 for the list of GRONs used in these examples

[0379] Table 15. List of GRONs used in this example

[0380] Methods

[0381] BFP transgenic Arabidopsis thaliana protoplasts derived from induced root tissue were seeded in a flat-bottomed 96-well plate at 250,000 cells per well at a cell density of 1x107 cells/ml. CRISPRS-encoded plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis U6 promoter, driving the sgRNA with a poly-T10 terminator. The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). CRISPRS plasmids along with GRON were introduced into protoplasts by PEG-mediated delivery at a final concentration of 0.05 μg/μL for CRISPRS, 0.16 μM for 41-mer GRONs. GRON treatments alone received a final GRON concentration after PEG delivery of 0.8 μM for the 41-mer. Protoplasts were incubated in the dark at 23°C for 72 hours, and then they were analyzed by flow cytometer in order to determine the percentage of GFP-positive protoplasts within a given treatment.

[0382] CRISPR consists of two components: the plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNA, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the BFP target gene. The spacer sequence of CRISPRS BFP is 5'CTCGTGACCACCTTCACCCA 3'. In this example, CRISPRS BFP was used, which targets the bfp gene. GRONs contain the non-coding sequence of the BFP gene near the conversion site. Table 16 shows a list of GRONs used

[0383] Table 16. List of GRONs used in this example.

[0384] Results

[0385] 41-mer 3PS GRONs are more effective than their unmodified Gron counterparts in mediating the conversion of BFP to GFP using CRISPR BFP (Figure 13).

[0386] Example 17: TALENS and GRONs in linen.

[0387] The purpose of this example is to demonstrate the conversion of EPSPS into flax in both 24 hours in protoplasts and 3 weeks after distribution in TALEN and GRONs plasmids. The TALENS used in this example target the epsps gene in the Linum usitatissimum genome by introducing into the shoot tip protoplast-derived plasmid(s) encoding TALENS, creating a double-strand break, and the GRON being used as a model for converting the epsps gene in a site-directed manner.

[0388] Methods

[0389] Flax protoplasts were isolated from shoots obtained from seedlings germinated in vitro. TALEN plasmids together with GRONs were introduced into PEG-mediated delivery protoplasts at a final concentration of 0.05 μg/μl and 0.5 μM respectively. Protoplasts were incubated in the dark at 25°C for up to 48 hours in liquid medium, or embedded in alginate beads (5 x 105 cells/ml) and cultured in liquid medium to induce cell division and microcalli formation. Protoplasts or microcalli samples obtained 24 h or 3 weeks after DNA delivery were analyzed by NGS to determine the percentage of cells (DNA reads) carrying the target mutation within a given treatment. The percentage of indels generated by imperfect NHEJ-mediated DNA repair was also estimated.

[0390] TALEN constructs include two arms (left and right), each consisting of a TAL effector-type DNA binding domain linked to a catalytic DNA cleavage Fokl domain. The TAL effector-like DNA-binding domain guides the TALEN arms to specific DNA sites that allow the FokI endonucleases from each arm to dimerize together and cleave double-stranded DNA. The encoded TALEN plasmids contain MASP: LuEPSPS_ (left arm) -T2A-LuEPSPS_ (right arm) with an rbcSE9 terminator. The sequence of LuEPSPS_ (left arm) is 5'TGGAACAGCTATGCGTCGTCCG 3' and the sequence of LuEPSPS_(right arm) is 5'TGAGTTGCCTCCAGCGGCT 3'. The targeting GRONs (144-mers) of LuEPSPS with or without flexible bases were used to determine its effect on the conversion rate.

[0391] Results

[0392] 24-hour protoplasts and 3-week-old microcalli have 0.067% and 0.051% EPSPS conversion, respectively, as determined by Next Generation Sequencing (Figure 14). Furthermore, these data show that TALEN is active and capable of cleaving the epsps target gene in Linum usitatissimum and forming indels of 2.60% and 1.84%, respectively, within 24 hours in protoplasts and up to 3 weeks in microcalli. . Furthermore, EPSPS conversion and indels are maintained up to 3 weeks after the TALEN and GRON plasmids are introduced.

[0393] Example 18: CRISPRs and GRONs in linen.

[0394] The purpose of this example is to demonstrate Cas9 activity in linen microcalli three and six weeks after delivery of a Cas9 plasmid. The CRISPRs used in this example target the Linum usitatissimum genome epsps gene by introducing into the shoot tip protoblast-derived plasmid(s) encoding the Cas9 gene and an sgRNAs. The sgRNA is a fusion of CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA guides Cas9 to target genes, where Cas9 creates a double-strand break in the epsps gene in a site-directed manner. Double-strand breaks in the epsps gene when repaired via the ubiquitous NHEJ pathway will cause indels to form around the cleavage site.

[0395] Methods

[0396] Flax protoplasts were isolated from shoots obtained from seedlings germinated in vitro. The encoded CRISPR plasmids contain the MAS promoter, driving the Cas9 coding sequence with an rbcSE9 terminator and Arabidopsis thaliana U6 promoter, driving the sgRNA with a poly-T10 terminator. CRISPRS plasmids were introduced into PEG-mediated delivery protoplasts to a final concentration of 0.05 μg/μL. Protoplasts were embedded in alginate beads (5 x 105 cells/mL), cultured in liquid medium, and incubated on a rotary shaker (30 rpm) in the dark at 25°C. Microcalli developed from single cells were analyzed by NGS, 3 and 6 weeks after CRISPRS plasmid delivery, to determine the percentage of cells (DNA reads) carrying indels generated through the NHEJ-mediated DNA repair pathway. prone to errors.

[0397] CRISPRS consists of two components: the plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNA, both of which were expressed from the same plasmid. The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA region contains the spacer sequence used to guide the Cas9 nuclease to the target gene. In this example, CRISPRS targets the epsps gene.

[0398] Results

[0399] 3- and 6-week-old Microcalli have 46.5% and 54.7% indel formation, respectively, as determined by Next Generation Sequencing (Figure 15). These data show that Cas9 is active and capable of cleaving the EPSPS target gene in Linum usitatissimum and forming indels. Furthermore, these indels are maintained up to 6 weeks after the CRISPR plasmid is introduced.

[0400] Example 19: Construction of designed nucleases

[0401] CRISPR-Cas

[0402] For construction of CRISPRS-Cas9 transient expression plasmids, a higher plant codon-optimized SpCas9 gene, containing an SV40 NLS, at both the N-terminus and C-terminus and a 2x FLAG tag at the N-terminus was synthesized as a series of GeneArt®™ chains (Life Technology, Carlsbad, CA), then cloned downstream of the mannopine synthase (MAS) promoter and upstream of the pea ribulose bisphosphate carboxylase (rbcsE9) terminator by the Gibson method . Next, an sgRNA cassette consisting of a chimeric gRNA whose expression is driven by the Arabidopsis U6 promoter, was synthesized as GeneArt® Strings™, then transported into the Cas9 containment construct using the Gibson method forming pBCRISPR. To specify the chimeric gRNA for the respective target sequence, pairs of DNA oligonucleotides encoding variable 20-nt sgRNA targeting sequences (Extended Data Table 2) were hybridized to generate short double-stranded fragments with 4-bp overhangs. . The fragments were ligated into Bbsl-digested pBCRISPR to produce CRISPR-Cas constructs BC-1, BC-2, and BC-3.

[0403] TALEN

[0404] The design and construction of TALEN BT-1 and LuET-1 expression constructs were rule-based, as described in Cermak et al., Nucleic Acids Res. 39, e82 (2011). The target sequence was selected based on the gene editing site and the i-residue repeat variable (RVD) following the rules that NG, HD, NI, and NN recognize T, C, A, and G, respectively. The TAL effector domain assembly linked to the heterodimeric FokI domains was completed through a commercial service (Geneart; Life Technologies). TALEN monomers were cloned downstream of the MAS promoter and upstream of the rbcE9 terminator using the Gibson method and expressed as a 2A coupled unit.

[0405] Cell culture and protoplast isolation

[0406] Surface-sterilized Arabidopsis seeds were germinated in % solid MS medium (minerals and vitamins according to XX; % concentrate; 87.7 mM sucrose) at 28 ° C under a 12 h light/dark cycle. Root materials from 2- to 3-week-old seedlings were collected and maintained in MS% liquid medium under low light conditions at 28°C. Root cultures were transferred and maintained in MSAR [0.22% 1/2 MS, 87.7 mM sucrose, 11.4 μM IAA, 2.3 μM 2,4-D, 1.5 μM 2iP, pH 5,8] three weeks before protoplast isolation. Roots were cut into approximately 6 mm segments and incubated in MSAP solution [0.22% 1/2 MS, 87.7 mM sucrose, 11.4 μM IAA, 2.3 μM 2,4-D, 1.5 μM 2iP, and 400 mM mannitol, pH 5.8] containing cell wall digestion enzymes [1.25% cellulase RS, 0.25% Macerozyme R-10, 0.6 M mannitol, MES 5 mM, 0.1% BSA] for 3-4 hours in the dark with gentle shaking. Released protoplasts were harvested and passed through a sterile 100 μm filter and 35 μm filter. The protoplast filtrate was mixed with 0.8 times the volume of Optiprep™ Density Gradient Medium (Sigma) and mixed gently. A solution of 60% W5 [154 mM NaCl, 5 mM KCl, 125 mM CaCl2-2H2O, 5 mM glucose, 10 mM MES, (pH 5.8)]/40% Optiprep followed by 90% W5 solution /10% Optiprep was slowly dipped into the filtrate/Optiprep solution to make a gradient, which was centrifuged at 198 RCF for 10 min. The white protoplast layer was collected and mixed with 2 times the volume of W5. Protoplasts were centrifuged at 44 RCF for 10 minutes and re-suspended in TM solution [14.8 mM MgCl2-6H2O, 5 mM MES, 572 mM mannitol, (pH 5.8)] at a density of 1x107 cells/ml. For experiments with zeocinTM (Life Technologies, Carlsbad, CA) and phleomycin (Invivogen, San Diego, CA), protoplasts were maintained in TM adjusted to pH 7.0 for 90 min on ice before transfection. For antibiotic concentrations see Extended Data in Figure 1.

[0407] Flax protoplasts were isolated from sprouts obtained from 3-week-old seedlings germinated in vitro. Shoot tips were finely minced with a scalpel, preplasmolyzed for 1 h at room temperature in B5 salts [B-form and vitamins (Gamborg et al., 1968), 4 mM CaCl2, 0.1 M glucose, 0 .3 M mannitol, 0.1 M glycine, 250 mg/L casein hydrolyzate, 10 mg/L L-cysteine-HCl, 0.5% polyvinylpyrrolidone (MW 10000), 0.1% BSA , 1 mg/l BAP, 0.2 mg/l NAA, and 0.5 mg/l 2,4-D], and incubated in a cell wall digestion enzyme solution containing medium-B supplemented with 0 .66% YC cellulase and 0.16% Macerozyme R-10 on a rotary shaker (50 rpm) at 25°C for 5 h. Released protoplasts were sieved and purified by density gradient centrifugation using OptiPrep layers (Sigma), counted with a hemocytometer, and held stationary overnight in the dark at a density of 0.5 x 106 protoplasts/ml in medium. B.

[0408] Protoplast transfection

[0409] In a 96-well flat-bottom plate, 2.5x10 cells per well were transfected with 10 pmol GRON, 10 pmol GRON plus 3.25 μg CRISPRS-cas or TALEN expression construct or mock of PEG [270 mM mannitol, 67.5 mM Ca (NO3)2, 38.4% PEG 1500, (pH 5.8)]. Cells and DNA were incubated with PEG on ice for 30 minutes followed by a wash with 200 μl of W5 solution. Finally, 85 μl of MSAP++ [MSAP containing 50 nM phytosulfokine-α and 20 μM n-propyl gallate] was added to 85 μl of MSAP and the cells were cultured in dim light conditions at 28°C.

[0410] After about 18 h of culture, the protoplasts were transfected with TALEN plasmid along with GRONs (20 μg of plasmid and 0.2 nmol of GRON/106 of protoplasts) using PEG-mediated delivery. Treated protoplasts were incubated in the dark at 25 °C for up to 48 h in medium B, or embedded in alginate beads 24 h after transfection, and cultured in KM-V liquid medium to induce cell division and microcalli formation. . For antibiotic experiments, 1.25x105 cells per well were transfected with 8 μM GRON CG13 using the PEG solution described above.

[0411] Cytometry Seventy-two hours after transfection, cells were analyzed by cytometry using the Attune® Acoustic Focus Cytometer (Applied Biosystems®) with excitation and emission detection, as appropriate for GFP. The background level was based on PEG-treated protoplasts without DNA delivery. For antibiotic experiments, protoplasts treated with zeocin or phleomycin before transfection were analyzed by cytometry 48 h after transfection.

[0412] Sequencing analysis

[0413] Genomic DNA was extracted from CRISPRS-cas or TALEN-treated protoplasts using the NucleoSpin® II Plant Kit, according to the manufacturer's recommendations (Machery-Nagel, Bethlehem, PA). Amplicons flanking the TALEN or CRISPRS target region were generated using Phusion® polymerase with 100 ng of genomic DNA and primers BFPF-1 (5'- GGACGACGGCAACTACAAGACC-3')/BFPR-1 (5'- TAAACGGCCACAAGTTCAGC-3') for Arabidopsis CRISPRS and TALEN; or LuEPF-1 (5'-GCATAGCAGTGAGCAGAAGC-3')/LuEPR-1 5'-AGAAGCTGAAAGGCTGGAAG-3' for L. usitatissimum from TALEN. Amplicons were purified and concentrated using Qiaquick MinElute columns (Qiagen, Valencia, CA). Deep sequencing of amplicons was performed by GeneWiz (South Plainfield, NJ) using a 2 × 250 bp MiSeq array (Illumina, San Diego, CA). For data analysis, read 1 and read 2 fastq files were imported into CLC Genomic Bench 7.0.4 (CLCBio, Boston, MA). Paired reads were merged into a single sequence if their sequences overlapped. A sequence for an amplicon was identified if the same or its reverse and complementary sequence contained both forward and reverse primary sequences. The occurrence of a unique sequence in a sample was recorded as its abundance. Percentage of indel or gene editing was calculated by dividing the number of reads with the edit or indel by the total number of reads sequenced, and multiplying by 100.

[0414] CRISPRS de-Cas photospacer sequence

[0415] TALEN binding domain sequences

[0416] GRON sequences used

[0417] Statistical analysis

[0418] Statistical significance was determined using a student's t-test, with a two-tailed distribution. P values <0.05 were considered significant. Data are presented as means and SEM.

[0419] Results

[0420] CRISPR-Cas nuclease activity and gene editing in A. thaliana protoplasts.

[0421] Engineered nucleases, such as TAL effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease Cas9 association (CRISPR-Cas9) can be programmed to target and cleave double-stranded DNA with high specificity. TALENS consists of two arms, both having a TAL effector-type DNA binding domain (TALE) linked to a catalytic DNA nuclease Fokl domain. TALE domains guide TALEN arms to specific DNA sites allowing dimerization of FokI endonucleases and subsequent generation of a double-strand break (DSB). The CRISPRS-Cas9 system consists of two components; a Streptococcus pyogenes nuclease of Cas9 (SpCas9) and a chimeric fusion of two RNAs (crRNA and tracrRNA) referred to as an engineered single guide (sgRNA). The sgRNA supports Cas9-targeted nucleic acid specificity through base pairing of its first 20 5' bases with the target DNA, subsequently resulting in a site-specific DSB.

[0422] In plants, DSBs are typically repaired by the error-prone nonhomologous end joining (NHEJ) DNA repair pathway, resulting in random deletions and/or insertions (indels) at the repair site. In contrast, precision genome editing relies on nuclease-induced DSBs near the targeted change to be repaired by homology-directed repair (HDR), a repair pathway that is more precise than NHEJ due to the requirement of a template DNA. homologous -in most cases, sister chromatids. To benefit from the HDR pathway, it is possible to use an engineered oligonucleotide as the template for editing DNA in a specific way, when cleaved by nucleases, or in a non-specific way, when used in combination with antibiotics that induce double-strand breaks. .

[0423] FIG. 16 depicts the nuclease activity of CRISPRS-Cas9 in Arabidopsis thaliana protoplasts derived from the BFP transgenic model system, in which a stably integrated BFP gene can be converted to GFP by editing the codon encoding H66 (CAC ^ TAC H66Y). When cells were treated with CRISPRS-Cas9 (BC-1), induced NHEJ indels were produced at a frequency of 0.79% near the H66 locus of the BFP gene by deep sequencing (Fig. 16a). Most indels were single bp and none longer than 9 bp. In contrast, cells treated with GRON alone or mock-treated did not exhibit indels (data not shown). These results show that the CRISPRS-Cas9 nuclease can actively target the BFP gene in this transgenic model system.

[0424] With regard to the efficacy of CRISPRS-Cas9 in combination with GRON to mediate BFP to GFP gene editing in protoplasts derived from our transgenic model system, a 7.4-fold improvement in BFP to GFP editing was observed when both CRISPRS-Cas9 and phosphorothioate (PS)-modified GRONs (CG6) are introduced simultaneously, when compared with GRON alone or CRISPRS-Cas9 alone treatments (Fig. 16b). These results demonstrate that the introduction of CRISPRS-Cas9 with PS-modified GRONs into Arabidopsis protoplasts significantly increases the frequency of BFP to GFP gene editing.

[0425] GRONs containing three adjacent PS modifications (herein referred to as 3PS) at both the 5' and 3' ends positively affect the editing of BFP to GFP when compared to an unmodified GRON. 3PS-modified GRON (CG2), when combined with CRISPR-Cas9 (BC-1), is more effective in editing BFP to GFP, when compared to an unmodified GRON model (CG1; Fig. 17a). Furthermore, a positive correlation between GRON editing and length was observed (Fig. 17b). Taken together, these results show that both modification and length of GRON can significantly improve the frequency of gene editing in plants such as Arabidopsis in the presence of CRISPR-Cas9.

[0426] When the GRON (CG6) modified by 3PS of nucleobase (nb) 201, or the GRONs (CG9) or (CG10) modified by 2'-O-methyl of nucleobase (nb) 201, which consist of the first ten bases of 5' RNA with the first RNA base or the first 9 RNA bases modified with 2'-O-methyl are introduced together with CRISPRS-Cas9 (BC-1) in Arabidopsis protoplasts no statistical difference in BFP editing for GFP was observed among them (Figure 17c). Similarly, when 3PS-modified GRON (CG3) of nb 201 or the Cy3-modified GRON (CG4) of nb 201, which comprises a 5' Cy3 and a 3' idC reverse base, were introduced together with CRISPRS -Cas9 (BC-3) stops in Arabidopsis protoplasts no statistical difference in editing frequencies was observed (Fig 17d). Overall, these data show that several modifications of GRON can significantly improve the frequency of gene editing in Arabidopsis in the presence of CRISPR-Cas9.

[0427] Based on these results with CRISPR-Cas9 and modified GRONs, it was determined whether modified GRONs together with TALEN pairs targeting the BFP gene also result in improved gene editing from BFP to GFP. To show the first effective nuclease activity at the BFP locus of Arabidopsis protoplasts were treated with TALENS (BT-1) and 0.51% of indels found at or near the cleavage site predicted by deep sequencing - indicating that TALENS are as actives such as CRISPRS-Cas9 in this exemplary system (Figure 18a). Most deletions were >10 bp but less than 50 bp, whereas insertions, significantly less abundant than deletions, were 3 bp or less. We next examined the efficacy of TALENS together with modified GRON for editing BFP to GFP. A 9.2-fold improvement in the editing frequency of BFP to GFP was observed when both TALEN BFP (BT-1) and GRON 3PS (CG7) are introduced when compared to GRON 3PS alone (Fig. 18b). Similar to the CRISPRS-cas experiments described above, these results demonstrate that the introduction of TALENS with 3PS-modified GRONs into Arabidopsis protoplasts also significantly increases the frequency of BFP to GFP gene editing.

[0428] The EPSPS (5'-enolpyruvylshikimate-3-phosphate synthase) loci in Linum usitatissimum (Common flax) was also used as a target in this system. The EPSPS genes encode an enzyme in the shikimate pathway that participates in the biosynthesis of aromatic amino acids phenylalanine, tyrosine, and tryptophan. In plants, EPSPS is a target for the herbicide, glyphosate, where it acts as a competitive inhibitor of the binding site for phosphoenolpyruvate.

[0429] TALENS targeting a location near two loci (T97I and P101A) in L. usitatissimum that when edited will result in glyphosate-tolerant EPSPS (Extended Data Fig. 18b) were selected. The distribution TALEN (LuET-1), together with a 5' Cy3-modified GRON (CG11) of nb 144 containing the targeted changes in T97I and P101A in protoplasts, the gene editing frequency of 0.19% in frequency of both loci and 0.5% indel seven days after introduction were observed (Fig. 18c, 18d). Most indels were 10 bp or less (Fig. 18c). These results demonstrate that the introduction of TALENS with Cy3-modified GRONs into L. usitatissimum protoplasts significantly increases the frequency of EPSPS gene editing and, furthermore, that multiple nucleotide edits can be performed with a single GRON.

[0430] Example 20: Effect of two members of the bleomycin family of antibiotics on conversion.

[0431] The objective of this series of examples was to evaluate the effect of antibiotics on conversion efficiencies.

[0432] Methods

[0433] Protoplasts from an Arabidopsis thaliana line with multiple copies of a blue fluorescent protein gene were treated with GRON as described in Example 1, with the following modification: before the addition of GRON, the protoplasts were kept for 90 minutes on ice in a TM solution (14.8 mM MgCl2 x 6H2O, 5 mM 2-(N-morpholino)ethanesulfonic acid, 572 mM mannitol), supplemented with 0, 250, or 1000 μg/ml zeocin™ or phleomycin. The pH of the solutions was adjusted to 7.0. The percentage of green fluorescent cells resulting from conversion of BFP to GFP was assessed by flow cytometry as described in Example 1.

[0434] Results

[0435] Zeocin and phleomycin at both concentrations used (250 and 1000 μg/ml) resulted in an increase in BFP gene editing for GFP (see Figure 19). Green fluorescent cells resulting from BFP to GFP gene editing were observed five days after GRON delivery (Figure 20).

[0436] References 9. LeCong et al 2013 Science: vol. 339 no. 6121pp. 819-823. 10. Jinek et al 2012 Science. 337:816-21 11. Wang et al 2008 RNA 14: 903-913 12. Zhang et al 2013. Plant Physiol. 161:20-27

[0437] Example 21: CRISPRs and GRONs in Rice Plant.

[0438] The objective of this experiment is to show the conversion of ACCase in Oryza sativa, 120 hours after the distribution of PEG from CRISPRS-Cas plasmids and GRONs into protoplasts. The CRISPRS-Cas used in this experiment targets the ACCase gene in the rice plant genome through introduction into protoplast plasmid(s) encoding the Cas9 gene and an sgRNAs. The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA guides the target genes of Cas9, where Cas9 creates a double-strand break in the ACCase gene and the GRON is used as a template to convert the ACCase gene in a site-directed manner.

[0439] Methods

[0440] rice plant protoplasts were isolated from calli. The CRISPRS-cas encoded plasmids contain the maize ubiquitin promoter driving the Cas9 coding sequence with an rbcSE9 terminator and a rice plant U6 promoter driving the sgRNA with a poly-T10 Future Terminator. CRISPRS-cas plasmids were introduced into PEG-mediated delivery protoplasts to a final concentration of 0.05 μg/μl. The GRONs with the following sequence, 5' VC TGA CCT GAA CTT GAT CTC AAT TAA CCC TTG CGG TTC CAG AAC ATT GCC TTT TGC AGT CCT CTC AGC ATA GCA CTC AAT GCG GTC TGG GTT TAT CTT GCT TCC AAC GAC AAC CCA AGC TCC CCC TCG TAG CTC TGC AGC CAT GGG AAT GTA GAC AAA GGC AGG CTG ATT GTA TGT CCT AAG GTT CTC AAT AAC CGA AGT GCC H 3 ', were used at a final concentration of 0.8 μM. Protoplasts were embedded in agarose (2.5 x 106 cells/mL), cultured in liquid medium, and incubated on a rotary shaker (60 rpm) in the dark at 28°C. Individual samples were analyzed by NGS, 120 hours after CRISPRS-cas plasmid and/or GRON delivery, to determine the percentage of cells (read DNA) carrying the ACCase conversion and having indels in the ACCase gene.

[0441] CRISPR consists of two components: the codon optimized plant Streptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which were expressed from the same plasmid. sgRNA is a fusion of CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA region contains the spacer sequence (5'- ACGAGGAGGGCTTGGGTTGTGG-3) used to guide the Cas9 nuclease to the target gene. In this experiment, CRISPRS targets the ACCase gene.

[0442] Results

[0443] At 120 hours, rice plant protoplasts have 0.026% ACCase conversion as determined by Next Generation sequencing. GRON alone controls without CRISPRS-Cas showed minimal conversion of 0.002% at 120 hours and treated controls showed no conversion. Furthermore, these data show that CRISPRS-cas is active and capable of cleaving the ACCase target gene and forming 8.0% indels.

[0444] References 13. LeCong et al 2013 Science: vol. 339 no. 6121 pp. 819-823. 14. Jinek et al 2012 Science. 337:816-21 15. Wang et al 2008 RNA 14: 903-913 16. Zhang et al 2013. Plant Physiol. 161:20-27

[0445] Example 22: CRISPRs and GRONs in Rice Plant

[0446] Summary: Targeted ACCase mutations were identified in 19-week-old calli by PCR and DNA sequencing analyses.

[0447] Introduction

[0448] The objective of this experiment is to show conversion of ACCase in Oryza sativa calli after delivery of PEG from CRISPRS-Cas plasmids and GRONs into protoplasts. The CRISPR-Cas used in this experiment targets the ACCase gene in the rice plant genome through the introduction of protoplast plasmid(s) encoding the Cas9 gene and an sgRNAs. sgRNA is a fusion of CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA guides Cas9 to the target gene, where Cas9 creates a double-strand break or a small nick at a target site in the ACCase gene and the GRONs are used as a template to convert the ACCase gene in a site-directed manner.

[0449] Results

[0450] The targeted OsACCase mutations described in the tables below were identified in 19-week-old calli by PCR and DNA sequencing analyses.

[0451] Methods

[0452] Protoplasts were isolated from suspension cultures initiated from seed-derived mature embryogenic calli. CRISPRS-cas plasmids with GRONs at a final concentration of 0.05 μg/μl and 0.8 μM, respectively, were introduced into protoplasts by PEG-mediated delivery method. An exemplary range for CRISPRS-cas plasmids at a final concentration includes, but is not limited to, 0.01 to 0.2 μg/μl. An exemplary range for GRON at a final concentration includes, but is not limited to, 0.01 to 4 μM. The CRISPRS-cas encoded plasmids contain the maize ubiquitin promoter driving the Cas9 coding sequence with an rbcSE9 terminator and a rice plant U6 promoter driving the sgRNA with a poly-T10 Future Terminator. Sequence information of the GRONs is described in Table 3. Following PEG treatment, protoplasts were embedded in agarose (1.25 x 106 cells/mL), cultured in liquid medium, and incubated on a rotary shaker (60 rpm) in the dark at 28°C. An exemplary range for embedding protoplasts in agarose includes, but is not limited to, 0.625 x 106 to 2.5 x 106 cells/ml. Samples from each treatment were analyzed by Next Generation Sequencing 4 weeks after CRISPR-Cas and/or GRON plasmid treatment to determine the proportion of cells (DNA reads) carrying ACCase conversion. Microcalli from converted treatments were released onto solid selection medium containing clethodime (0.25-0.5 μM) or sethoxydime (2.5 μM). Individual calli lines growing in this selection medium after 19 weeks of culture were analyzed by internal screening methods as well as DNA sequencing in order to identify individual calli containing the targeted ACCase conversions.

[0453] CRISPRS consists of two components: Cas9, such as plant codon-optimized Cas9 (SpCas9) from Streptococcus pyogenes, and sgRNA, both of which were expressed from plasmid(s). Cas9 sgRNA and can also be delivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA region contains the spacer sequences described in Table 2, which were used to guide the Cas9 nuclease to the target gene. In this experiment, CRISPRS targets the rice plant ACCase gene.

[0454] List of conversions in OsACCase at two different sites within the gene, site 1 and site 2. For each site, all combinations of conversion events are possible.

[0455] List of CRISPR-Cas gRNA spacer sequences used in this experiment. Spacer length can vary up to ±20 bp. Mismatches within the spacer sequence can be tolerated up to 10 bp.

[0456] A list of GRON sequences suitable for use in this experiment is provided in the table below (V= CY3; H= 3'DMT dC CPG).

[0457] Example 23: CRISPRS and GRONs in Linen

[0458] Summary: Targeted LuEPSPS mutations were identified in four-week-old calli by PCR and DNA sequencing analyses.

[0459] Introduction

[0460] The objective of this experiment is to show conversion of EPSPS genes in the Linum usitatissimum genome into shoot tip-derived protoplasts by PEG-mediated delivery of CRISPRS-Cas and GRONs plasmids. The CRISPRS-cas and GRONs used in this experiment target the EPSPS genes in flax genome. CRISPRS consists of two components: a Cas9, such as a plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9), and sgRNA, both of which are expressed from plasmid(s). Cas9 sgRNA and can also be delivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA guides Cas9 to targeted genes, where Cas9 creates a double-strand break or short nick in the EPSPS genes and the GRONs are used as a template to convert EPSPS genes in a site-directed manner.

[0461] Results

[0462] Targeted LuEPSPS mutations (T97I and/or P101A, P101S or P101T and/or G96A) were identified in four-week-old calli by PCR and DNA sequencing analyses. Shoots were regenerated from the converted calli.

[0463] Methods

[0464] Flax protoplasts were isolated from shoots obtained from seedlings germinated in vitro. The encoded CRISPRS-Cas plasmids contain the MAS promoter, which drives the Cas9 coding sequence with an rbcSE9 terminator, and the Arabidopsis thaliana U6 promoter, which drives the sgRNA with a poly-T10 terminator. CRISPRS-cas plasmids were introduced into PEG-mediated delivery protoplasts to a final concentration of 0.05 μg/μl. GRONs targeting each of the two flax LuEPSPS genes (Table 2) were used at a final concentration of 4.0 μM. An exemplary range for CRISPR-Cas plasmids at a final concentration includes, but is not limited to, 0.01 to 0.2 μg/μl. An exemplary range for GRON at a final concentration includes, but is not limited to, 0.01 to 4 μM. Protoplasts were embedded in alginate beads (5 x 105 cells/ml), cultured in liquid medium, and incubated on a rotary shaker (30 rpm) in the dark at 25°C. An exemplary range for incorporating protoplasts into alginate beads includes, but is not limited to, 3.0 x 105 to 7.5 x 105 cells/ml. Microcalli developed from individual cells were analyzed by Next Generation Sequencing, 3 and 7 weeks after CRISPRS-cas and GRON plasmid delivery, to determine the proportion of cells (DNA reads) that carry the specific mutations in the genes. LuEPSPS. Larger calli were grown from converted 8-week-old microcalli plated on solid regeneration medium, and shoots began to differentiate from regenerated calli after about 4-8 weeks. Converted calli and shoots with the EPSPS gene mutations were identified by PCR and DNA sequencing analyses.

[0465] CRISPRS consists of two components: Cas9 (SpCas9) and plant codon-optimized Streptococcus pyogenes sgRNA, both of which were expressed from plasmid(s). The sgRNA is a fusion of CRISPRS RNA (crRNA) and transactivation crRNA (tracrRNA). The crRNA region contains the spacer with sequences described in the table below, which were used to guide the Cas9 nuclease to the EPSPS targeted genes.

[0466] List of CRISPRS-cas gRNA spacer sequences used in this experiment. The spacer length can vary up to ±20 bp. Mismatches within the spacer sequence can be tolerated up to 10 bp.

[0467] A list of GRON sequences suitable for use in this experiment is provided in the table below (V= CY3; H= 3'DMT dC CPG).

[0468] One skilled in the art easily recognizes that the present description is well adapted to achieve the objectives and obtain the mentioned ends and advantages, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended to be limitations on the scope of the disclosure.

[0469] It will be immediately apparent to an individual skilled in the art that variant substitutions and modifications may be made to the disclosure disclosed herein without deviating from the scope and spirit of the disclosure.

[0470] The disclosure illustratively described in this document may be properly practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed in this document. Thus, for example, in each instance herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be substituted for any of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that the use of such terms and expressions will exclude any equivalents of the features shown and described or parts thereof, but it is recognized that various Modifications are possible within the scope of the claimed disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts disclosed herein may be relied upon by those skilled in the art, and that such modifications and variations are considered to be being within the scope of the present disclosure, as defined by the added claims.

[0471] Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement, and variation of the disclosed disclosures may be relied upon by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples presented herein are representative of preferred embodiments, are exemplary, and are not intended to be limitations on the scope of the disclosure.

[0472] Disclosure has been described extensively and generically here. Each of the narrower species and subgeneric groups that fall within the generic disclosure are also part of the disclosure. This includes generic description of the disclosure with a disclaimer or negative limitation, removing any such matter, regardless of whether or not the removed material is specifically recited herein.

[0473] Furthermore, where characteristics or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is thereby also described in terms of any individual member or subgroup of members of the group Markush.

[0474] All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, this specification, including definitions, will serve as the basis for control.

[0475] Other embodiments are set forth in the following claims.

Claims (11)

1. Method for causing a genetic change in a plant cell, characterized by the fact that it comprises exposing said cell to a DNA cutter and a gene repair oligonucleobase (GRON), wherein the DNA cutter is CRISPR-Cas9 , wherein the GRON includes one or more changes from conventional RNA and DNA nucleotides, said changes being selected from one or more 2'O-methyl nucleotides at the 5' and/or 3' ends, a reverse base (idC ) at the 3' end, an O-(2-methoxyethyl) (MOE) modification at the 5' and/or 3' ends, one or more Cy3 groups at the 5' and/or 3' ends, where Cy3 denotes a blocked phosphoramidite which upon incorporation into an oligonucleotide produces a 3,3,3',3'-tetramethyl N,N'-isopropyl substituted by an indomonocarbocyanine dye, and one or more 3PS groups at the 5' and/or 3' ends, where 3PS denotes 3 phosphothioate bonds, and wherein said GRON is between 15 and 210 nucleotides in length. 2. Method according to claim 1, characterized by the fact that said GRON is single-stranded. 3. Method according to claim 1 or 2, characterized by the fact that the GRON has an oscillating base pair in relation to the target sequence for the genetic alteration. 4. Method according to any one of claims 1 to 3, characterized in that said plant is selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, green corn, wheat, barley , rice, alfalfa, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, onion, soybean, soybean spp, sugar cane, pea, chickpea, field pea, broad beans, lentils, turnips, rutabaga, Brussels sprouts, lupins, cauliflower, cabbage, broad beans, poplar, pine, eucalyptus, grapes, citrus, triticale, alfalfa, rye, oats, grass and forage grasses, flax, rapeseed , mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, cassava, lily and nut-bearing plants. 5. Method according to any one of claims 1 to 4, characterized in that said plant is canola, or in which said plant is corn, or in which said plant is green corn, or in which said plant is wheat, or wherein said plant is rice, or wherein said plant is sorghum, or wherein said plant is potato, or wherein said plant is soybeans, or wherein said plant is flax, or wherein said plant is flax, or wherein said plant is said plant is rapeseed, or wherein said plant is cassava, or wherein said plant is sunflower. 6. Method according to any one of claims 1 to 5, characterized by the fact that said plant is a plant of the genus Brassica selected from B. carinata, B. elongate, B. fruticulosa, B. juncea, B. napus, B. narinosa, B. nigra, B. oleracea, B. perviridis, B. rapa (syn B. campestris), B. rupestris, B. septiceps and B. tournefortii, in particular where the plant is B. carinata. 7. Method according to any one of claims 1 to 6, characterized by the fact that multiple genetic changes are made. 8. Method according to any one of claims 1 to 7, characterized in that two or more guide RNAs are used. 9. Method according to claim 8, characterized by the fact that each of the more than one guide RNAs is complementary to a different target for genetic alteration. 10. Method according to any one of claims 1 to 9, characterized by the fact that CRISPR-Cas9 includes a nickase. 11. Method according to any one of claims 1 to 10, characterized by the fact that CRISPR-Cas9 includes two or more nickases, and/or in which two or more nickase cutters are on opposite strands of the target nucleic acid sequence, or wherein two or more nickase cutters are on the same strand of the target nucleic acid sequence.