Herbicide resistance genes for mitochondrial transformation

Abstract

This disclosure relates to genetically modified cells containing mitochondria transformed with polynucleotides encoding herbicide-resistant enzymes, so that they can grow in the presence of an inhibitor of said herbicide-resistant enzymes.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 451,173, filed Mar. 9, 2023, which is hereby incorporated by reference in its entirety.

[0002] Statement regarding federally sponsored research This invention was made with government support under Contract No. 58-5062-0-001 awarded by the United States Department of Agriculture (USDA) in a Cooperative Research and Development Agreement (CRADA). The government has certain rights in this invention.

[0003] Incorporation of Sequence Listing by Reference This application is filed with an electronic sequence listing. The sequence listing is provided as file name 51090-707.601_SL.xml created on Mar. 1, 2024 and is 190,856 bytes in size. The electronic form information of the sequence listing is hereby incorporated by reference in its entirety.

Background Art

[0004] Modification of the mitochondrial genome is very important in basic research and applied research. Transgenic plants with a stably modified mitochondrial genome can have new traits such as herbicide tolerance, insect resistance, and / or accumulation of valuable proteins such as pharmaceutical proteins and industrial enzymes.

Summary of the Invention

[0005] This specification discloses a method for transforming the mitochondria of a cell, comprising: (a) introducing a first polynucleotide encoding a first polypeptide into the mitochondria of the cell, wherein the cell is a plant cell or an algal cell, the first polypeptide encoding the first polypeptide is a variant of a naturally occurring polypeptide, the naturally occurring polypeptide comprises an enzyme activity inhibited by a herbicide, and the variant of the naturally occurring polypeptide comprises an enzyme activity that exhibits resistance to the herbicide; (b) growing the cell under conditions in which the first polypeptide is expressed; (c) growing the cell in a medium in which a herbicide is present at an effective concentration; and (d) selecting a transformed cell comprising transformed mitochondria containing the first polynucleotide. In some embodiments, this specification discloses cells produced by the method described herein, which are plant cells selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, cotton cells, and soybean cells. In some embodiments, plants, cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, produced from plant cells described herein, are disclosed, and these cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, include an edited mitochondrial genome.

[0006] This specification describes a method for transforming the mitochondria of a cell, comprising: (a) a first polynucleotide encoding a first polypeptide, wherein the first polypeptide is a variant of a naturally occurring polypeptide, the naturally occurring polypeptide having enzyme activity inhibited by a herbicide, and the variant of the naturally occurring polypeptide having enzyme activity that exhibits resistance to the herbicide; and (ii) a second polynucleotide encoding a selectable marker, wherein the selectable marker enables the cell to grow in the presence of a selectant. A method is disclosed comprising the steps of: (b) introducing a second polynucleotide in which the second polynucleotide does not encode a first polypeptide; (c) growing cells under conditions in which a selectable marker is expressed; (d) growing cells in a medium containing a selector for the selectable marker, wherein the selector is present at an effective concentration; and (e) selecting transformed cells containing transformed mitochondria, wherein the transformed mitochondria contain a first polynucleotide, and further, the transformed cells containing the transformed mitochondria can be grown in a medium containing a herbicide, wherein the herbicide is present at an effective concentration. In some embodiments, cells produced by the method described herein are disclosed, which are plant cells selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, cotton cells, and soybean cells. In some embodiments, plants, cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, produced from plant cells described herein, and cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, include an edited mitochondrial genome.

[0007] This specification describes a method for controlling weeds, comprising the step of growing a plurality of plants in the presence of a herbicide that is a plant enzyme inhibitor, wherein at least one of the plurality of plants contains mitochondria containing heterologous polynucleotides encoding a variant of the plant enzyme, the variant of the plant enzyme has enzymatic activity that exhibits resistance to the herbicide, and the presence of the herbicide is sufficient to selectively promote the growth of at least one of the plurality of plants, resulting in increased growth of at least one of the plurality of plants compared to plants lacking the heterologous polynucleotide. In some embodiments, this specification describes transgenic plants or parts thereof containing the cells described herein. In some embodiments, fields or greenhouses may include the transgenic plants or parts thereof described herein. In some embodiments, food may include the cells described herein. In some embodiments, fields may include the cells described herein. In some embodiments, kits may include the cells or transgenic plants or parts thereof described herein.

[0008] This specification describes cells comprising an edited mitochondrial genome, wherein the cells are plant cells or algal cells, and the edited mitochondrial genome comprises heterologous polynucleotides encoding variants of naturally occurring polypeptides having enzymatic activity, wherein the enzymatic activity of the naturally occurring polypeptide is inhibited by a herbicide, and the enzymatic activity of the variant of the naturally occurring polypeptide having enzymatic activity exhibits resistance to the herbicide. In some embodiments, this specification describes transgenic plants or parts thereof comprising the cells described herein. In some embodiments, fields or greenhouses may include the transgenic plants or parts thereof described herein. In some embodiments, food may include the cells described herein. In some embodiments, fields may include the cells described herein. In some embodiments, kits may include the cells or transgenic plants or parts thereof described herein.

[0009] Reference All publications, patents, and patent applications referenced herein are cited herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually designated to be cited by reference. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows a map of plasmid pNAP170. This plasmid contains three nuclear expression cassettes: pUBI1::MTS-ALS(HR-LS)::OCS terminator, pACT1::MTS-ALS(SS)::NOS terminator, and p35S::HPT::CaMV terminator. [Figure 2] This figure shows the growth of rice callus cells in a culture medium containing chlorsulfuron, with the rice callus cells transformed with pNAP170. The callus cells within the circles were subjected to further selection. [Figure 3] This figure shows a map of plasmid pNAP198. This plasmid contains an mALS(HR-LS) coding region operably linked to a hybrid T7+ rice ATP1 promoter and a hybrid T7+ rice ATP1 terminator. The plasmid also contains an eGFP coding sequence operably linked to a rice COB1 promoter and the 5'UTR and rice COB1 terminator. Furthermore, the plasmid contains a B4 element associated with autonomous replication in rice mitochondria. [Figure 4] This figure shows the growth of rice callus cells in a culture medium containing chlorsulfuron, where the rice callus cells were simultaneously transformed with pNAP195 and pNAP198. Callus samples within the circle were subjected to further selection. [Figure 5]This figure shows a map of plasmid pNAP432. This plasmid contains donor DNA used to transform rice mitochondria. The donor DNA has an mALS (HR-LS) coding region (with nad4L RNA editing sites) that is operably ligated to a hybrid T7+ rice ATP1 promoter and a cleaved hybrid T7+ rice ATP1 terminator. The donor DNA has a 1.6kb rice mitochondrial DNA homology region at the 5' end and a 1.2kb region at the 3' end. [Figure 6A] This figure shows the growth of transformed rice callus cells in a culture medium containing chlorsulfuron. Figure 6A shows rice callus cells simultaneously transformed with donor DNA isolated from plasmids pNAP195 and pNAP432. [Figure 6B] This figure shows the growth of transformed rice callus cells in a culture medium containing chlorsulfuron. Figure 6B shows rice callus cells simultaneously transformed with donor DNA isolated from plasmids pNAP195 and pNAP433. [Figure 7A] This figure shows the effect of disulfiram on the inhibition of wild-type rice callus growth by oligomycin. Figure 7A shows rice callus on day 0. Plates 1-4 have ND2 medium as the basic medium with the additives shown. Plate 1: 0.5% sucrose. Plate 2: 0.5% sucrose and 100 μM disulfiram. Plate 3: 0.5% sucrose and 1 mg / L oligomycin. Plate 4: 0.5% sucrose and 100 μM disulfiram and 1 mg / L oligomycin. Figures 7A and 7B show that disulfiram may increase the ability of oligomycin to inhibit wild-type rice callus growth in the presence of 0.5% sucrose. [Figure 7B]This figure shows the effect of disulfiram on the inhibition of wild-type rice callus growth by oligomycin. Figure 7B shows rice callus on day 14. Plates 1-4 have ND2 medium as the basic medium with the additives shown. Plate 1: 0.5% sucrose. Plate 2: 0.5% sucrose and 100 μM disulfiram. Plate 3: 0.5% sucrose and 1 mg / L oligomycin. Plate 4: 0.5% sucrose and 100 μM disulfiram and 1 mg / L oligomycin. Figures 7A and 7B show that disulfiram may increase the ability of oligomycin to inhibit wild-type rice callus growth in the presence of 0.5% sucrose. [Figure 8] This figure shows the PCR analysis of the 5' and 3' integration sites of donor DNA containing sulfonylurea resistance genes in rice mitochondrial DNA. The events analyzed here were initially transformed simultaneously with pNAP432 donor DNA fragments and pNAP195 plasmid DNA. The integration sites were amplified, with one primer complementary only to rice mitochondrial DNA and the other primer complementary only to donor DNA (Example 11). Samples from the second PCR of the nested PCR reaction were separated on the gel. Lanes 1-8: 5' junction amplification. Lanes 9-15: 3' junction amplification. Lanes 1-15 correspond to PCR analysis of the following samples: #1: HH43 event (regenerated leaf), #2: wild-type leaf, #3: HH43 event A callus, #4: HH43 event B callus, #5: no DNA control, #6: wild-type rice callus, #7: event T033 callus, #8: blank lane (no sample load). The arrows on the left and right sides of the gel indicate the expected positions of the 5' junction fragment (1821 bp) and 3' junction fragment (1432 bp), respectively. Lane M contains molecular size standards. [Figure 9]This figure shows the PCR analysis of the 5' integration site of glyphosate-resistant donor DNA in rice mitochondrial DNA. The events analyzed here were first transformed with a pNAP661 donor DNA fragment. The integration site was amplified, with one primer complementary only to rice mitochondrial DNA and the other primer complementary only to donor DNA (Example 12). Lanes 1-16 correspond to PCR analysis of events TT53-TT67 and callus tissue from wild-type callus, respectively. Lane M contains a molecular size standard. Arrows indicate the position of the predicted 5' junction fragment. Events TT57 and TT58 produced PCR fragments of the predicted size (1.8kb) for 5' integration at the target site. [Figure 10] This figure shows the PCR analysis of the 3' integration site of glyphosate-resistant donor DNA in rice mitochondrial DNA. The events analyzed here were first transformed with a pNAP661 donor DNA fragment. The integration site was amplified, with one primer complementary to rice mitochondrial DNA only and the other primer complementary to donor DNA only (Example 12). Lanes 1-15 correspond to the PCR analysis of callus tissue from events TT51, TT57, TT58, TT13, TT14, TT17, TT25, TT31, TT33, TT41, TT42, TT46, TT61, TT64, and TT67, respectively. Lane 16: wild-type rice callus. Lane 17: no DNA control. Lane M contains a molecular size standard. Arrows indicate the location of 1.4kb 3' junction fragments from events TT31 and TT46. This 3' junction fragment was approximately 0.3 kb shorter than expected, as confirmed by subsequent DNA sequencing (Example 12). [Figure 11]This figure shows the PCR analysis of the 5' integration site of glufosinate-resistant donor DNA in rice mitochondrial DNA. The events analyzed here were first transformed with a cleaved CsiI-BmtI donor DNA fragment derived from pNAP643. The integration site was amplified, with one primer complementary only to rice mitochondrial DNA and the other primer complementary only to the cleaved donor DNA fragment (Example 13). Lanes 1-18 correspond to the PCR analysis of callus tissue from events S94-S111, respectively. Lane M contains a molecular size standard. Arrows indicate the predicted location of the 5' junction fragment. Events S96, S109, and S111 produced PCR fragments of the predicted size (1.8kb) for 5' integration at the target site. [Figure 12] This figure shows the PCR analysis of the 3' integration site of glufosinate-resistant donor DNA in rice mitochondrial DNA. The events analyzed here were first transformed with a cleaved CsiI-BmtI donor DNA fragment derived from pNAP643. The integration site was amplified, with one primer complementary to mitochondrial DNA only and the other primer complementary to donor DNA only (Example 13). Lanes 1-18 correspond to the PCR analysis of callus tissue from events S94-S111, respectively. Lane M contains a molecular size standard. The arrows indicate the location of 1.4kb 3' junction fragments from events S96 and S109. These 3' junction fragments were approximately 0.3kb shorter than expected, as confirmed by subsequent DNA sequencing (Example 13). [Modes for carrying out the invention]

[0011] In some cases, mitochondrial genome editing can be more difficult than nuclear genome or plastid genome editing. In some cases, novel selectable marker genes can be used to generate and identify cells containing the edited mitochondrial genome. In some cases, novel selectable marker genes may be required to edit the mitochondrial genome of plants.

[0012] In some embodiments herein, methods and compositions for constructing and using organisms comprising polynucleotides encoding polypeptides, wherein the polypeptides are variants of naturally occurring polypeptides having enzymatic activity. In some embodiments, the enzymatic activity of naturally occurring polypeptides can play a crucial role in plant growth, development, or survival. In some embodiments, the enzymatic activity of naturally occurring polypeptides is inhibited by one or more herbicides. In some embodiments, the enzymatic activity of naturally occurring polypeptides may include acetolactate synthase (ALS) activity, 5-enol-pyruvir-schimate-3-phosphate synthase (EPSPS) activity, and glutamine synthetase (GS) activity. Table 1 shows non-limiting examples of herbicides and their corresponding targets. In some embodiments, the enzymes may originate from bacteria or eukaryotes. In some embodiments, variants of naturally occurring enzymes described herein (e.g., ALS, EPSPS, or GS) may exhibit resistance to herbicides.

[0013] In some cases, the enzymatic activity of naturally occurring polypeptides is acetolactate synthase activity, and herbicides are inhibitors of acetolactate synthase (e.g., sulfonylurea or imidazolinone). In some embodiments, polynucleotides can encode an enzyme having acetolactate synthase (ALS) activity or a bioactive fragment thereof. In some embodiments, the enzyme may be the herbicide-resistant acetolactate synthase large subunit (ALS-LS) polypeptide of Oryza sativa or a bioactive fragment thereof. In some embodiments, the terms "ALS-LS," "ALS(LS)," "ALS large subunit," and "ALS catalytic subunit" may be used interchangeably. In some embodiments, herbicide-resistant ALS-LS or a bioactive fragment thereof in mitochondria can enable growth in the presence of an inhibitor of ALS, which may be made available as a selectable marker. In some embodiments, the herbicide-resistant ALS-LS polypeptide disclosed herein may include the sequence shown in SEQ ID NO: 7. In some embodiments, the herbicide-resistant ALS-LS polypeptides disclosed herein may contain at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 7.

[0014] In some cases, the enzymatic activity of a naturally occurring polypeptide is 5-enolpyruvir schimate-3-phosphate synthase (EPSPS) activity, and the herbicide is an inhibitor of EPSPS (e.g., glyphosate). In some embodiments, a polynucleotide can encode an enzyme having EPSPS activity or a biologically active fragment thereof. In some embodiments, the enzyme may be herbicide-resistant EPSPS activity or a biologically active fragment thereof. In some embodiments, herbicide-resistant EPSPS in mitochondria or a biologically active fragment thereof can enable growth in the presence of an EPSPS inhibitor, which may be made available as a selectable marker.

[0015] In some cases, the enzymatic activity of the naturally occurring polypeptide is glutamine synthetase (GS) activity, and the herbicide is an inhibitor of GS (e.g., glufosinate). In some embodiments, the polynucleotide can encode an enzyme having GS activity or a biologically active fragment thereof. In some embodiments, the enzyme can be a herbicide-tolerant glutamine synthetase polypeptide or a biologically active fragment thereof. In some embodiments, the herbicide-tolerant GS or its biologically active fragment in mitochondria can enable growth in the presence of an inhibitor of GS that can be used as a selectable marker. In some embodiments, the transformed mitochondria can contain a polynucleotide. In some embodiments, the transformed mitochondria can contain an edited mitochondrial genome.

[0016] [Table 1]

[0017] This specification further discloses a method for transforming mitochondria, the method comprising: (a) introducing into the mitochondria of a cell a first polynucleotide encoding a first polypeptide, wherein the first polypeptide is a variant of a naturally occurring polypeptide having enzymatic activity, the enzymatic activity of the naturally occurring polypeptide can be inhibited by a herbicide, and the enzymatic activity of the variant of the naturally occurring polypeptide can exhibit resistance to the herbicide; (b) growing the cell under conditions in which the first polypeptide is expressed; and (c) growing the cell in a medium in which the herbicide is present at an effective concentration. In some embodiments, a method for selecting or screening for transformed mitochondria comprising a gene modification event, e.g., an edited mitochondrial genome expressing a polynucleotide encoding a polypeptide described herein, after introduction into an organelle (e.g., nucleus, mitochondria), cell, or tissue of interest. In some embodiments herein, recombinant plant cells, recombinant plant tissues, transgenic plants, transgenic plant seeds, transgenic plant roots, transgenic plant flowers, transgenic plant fruits, transgenic plant pollen, and transgenic plant progeny comprising an edited mitochondrial genome described herein are disclosed. Optionally, transgenic plants comprising an edited mitochondrial genome described herein can exhibit resistance to killing and / or growth inhibition by one or more herbicides. In some embodiments, transgenic seeds and transgenic progeny plants of a parental transgenic plant comprising an edited mitochondrial genome described herein are used to produce food, feed, industrial products, oils, nutritional foods, and other valuable products. Optionally, the methods and compositions described herein can be used to control the growth of unwanted plants among crops or other plants comprising the edited mitochondria described herein, thereby improving the growth and production of the crop or other plant of interest.

[0018] Definitions In some embodiments, the meanings of the abbreviations can be as follows: "sec" can mean seconds, "min" can mean minutes, "h" can mean hours, "d" can mean days, "μL" can mean microliters, "ml" can mean milliliters, "L" can mean liters, "μM" can mean micromoles, "mM" can mean millimoles, "M" can mean moles, "mmol" can mean millimoles, "μmol" can mean micromoles, "g" can mean grams, "μg" can mean micrograms, "ng" can mean nanograms, "U" can mean units, "nt" can mean nucleotides, "bp" can mean base pairs, "kb" can mean kilobases, and "kbp" can mean kilobase pairs.

[0019] In some embodiments, “transgenic” can refer to any cell, cell line, callus, tissue, part of an organism, or whole organism (e.g., a plant) whose genome has been edited or modified by the presence of heterologous or exogenous nucleic acids, such as recombinant DNA constructs. In some embodiments, transgenic events may include those produced by sexual mating or asexual reproduction. In some embodiments, the term “transgenic” may not include edited genomes or genome modifications (e.g., chromosomal or extrachromosomal) caused by breeding methods or by naturally occurring events such as random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. In some embodiments, the term “transgenic” may include edited genomes or genome modifications (e.g., chromosomal or extrachromosomal) caused by breeding methods or by naturally occurring events such as random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0020] In some embodiments, the “genome” of a cell or an entire organism may include chromosomal DNA found in the nucleus (nuclear DNA) and DNA found in cytoplasmic organelles (e.g., mitochondrial DNA, plastid DNA). The methods and compositions of this disclosure can be used to edit the genome of the nucleus, cytoplasmic organelles (e.g., mitochondria, plastids), or any combination thereof.

[0021] In some embodiments, the terms “complete complement” and “full-length complement” may be used interchangeably herein and may refer to the complement of a given nucleotide sequence. In some embodiments, the complement and the nucleotide sequence may contain the same number of nucleotides. In some embodiments, the complement and the nucleotide sequence may contain 100% complementarity. In some embodiments, the complement and the nucleotide sequence may have different numbers of nucleotides. In some embodiments, complementarity (e.g., between complement and nucleotide sequences) may be at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some embodiments, the complementarity (e.g., between complement and nucleotide sequences) may be up to approximately 10%, up to approximately 15%, up to approximately 20%, up to approximately 25%, up to approximately 30%, up to approximately 35%, up to approximately 40%, up to approximately 45%, up to approximately 50%, up to approximately 55%, up to approximately 60%, up to approximately 65%, up to approximately 70%, up to approximately 75%, up to approximately 80%, up to approximately 85%, up to approximately 90%, up to approximately 91%, up to approximately 92%, up to approximately 93%, up to approximately 94%, up to approximately 95%, up to approximately 96%, up to approximately 97%, up to approximately 98%, up to approximately 99%, or 100%.

[0022] In some embodiments, “polynucleotide,” “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are interchangeable and may refer to polymers of nucleic acids (e.g., RNA, DNA, or both, and their analogues) that are single-stranded or double-stranded (or both single-stranded and double-stranded) and optionally contain synthetic, non-natural, or modified nucleotide bases. In some embodiments, nucleotides (e.g., in their 5'-monophosphate form) may be referred to by single-letter designations (for RNA or DNA, respectively) as follows: "A" is an adenylate or deoxyadenylate, "C" is a citidylate or deoxycytidylate, "G" is a guanylate or deoxyguanylate, "U" is a uridilate, "T" is a deoxythymidylate, "R" is a purine-based nucleotide (A or G), "Y" is a pyrimidine-based nucleotide (C or T), "K" is G or T, "H" is A or C, "I" is inosine, and "N" is any nucleotide. In some embodiments, the polynucleotide may be linear or cyclic.

[0023] In some embodiments, as used herein, “nucleic acid” may refer to a polynucleotide sequence or a fragment thereof. In some embodiments, nucleic acid may comprise nucleotides. In some embodiments, nucleic acid may exist in a cell-free environment. In some embodiments, nucleic acid may be a gene or a fragment thereof. In some embodiments, nucleic acid may be DNA. In some embodiments, nucleic acid may be RNA. In some embodiments, nucleic acid may comprise one or more analogues (e.g., a modified skeleton, sugar, or nucleic acid base). In some embodiments, non-limiting examples of analogues include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosin, and waiosin.

[0024] In some embodiments, “polypeptide,” “peptide,” “amino acid sequence,” and “protein” are interchangeable herein and may refer to polymers of amino acid residues. In some embodiments, these terms may apply to amino acid polymers and / or naturally occurring amino acid polymers in which one or more amino acid residues may be artificial chemical analogs of, for example, corresponding naturally occurring amino acids. In some embodiments, the terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may include modifications including, but not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues. In some embodiments, an enzymatic polypeptide may include an active site that enhances the enzymatic activity. In some embodiments, the active site of an enzymatic polypeptide may include a substrate-binding domain and a catalytic domain that catalyze the reaction of the substrate.

[0025] In some embodiments, a “functional fragment” of a polynucleotide or polypeptide may refer to any subset of a sequence of nucleotides or amino acids, respectively, where the original (e.g., wild-type) activity (or substantially similar activity) of the polynucleotide or polypeptide may be preserved. In some embodiments, the terms “functional fragment,” “functional sub-fragment,” “functionally equivalent fragment,” “functionally equivalent sub-fragment,” “functionally equivalent fragment,” “biologically active fragment,” and “functionally equivalent sub-fragment” may be used interchangeably herein.

[0026] In some embodiments, the term “effective concentration” of a herbicide or selective agent to a plant, callus, cell, or other plant tissue may be the amount that causes a reduction in the growth rate, cessation of growth, or death of the plant, callus, cell, or other plant tissue compared to a plant, callus, cell, or other plant tissue that has not been exposed to the herbicide or selective agent. The effective concentration may allow for the distinction between samples that exhibit tolerance or resistance to the herbicide or selective agent and those that do not.

[0027] In some embodiments, the terms “functional variant,” “functionally equivalent variant,” and “functionally equivalent variant” may be used interchangeably herein. In some embodiments, in the context of polynucleotides or polypeptides, these terms may refer to variants of nucleic acid sequences or amino acid sequences, respectively, where the original activity (or substantially similar activity) of the polynucleotide or polypeptide may be retained. In some embodiments, fragments and variants may be obtained by methods such as site-directed mutagenesis and synthetic construction.

[0028] In some embodiments, the activity of the functional fragment or functional variant may be, for example, about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 10% of the original (e.g., wild-type) activity.

[0029] In some embodiments, “RNA transcript” may refer to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. In some embodiments, an RNA transcript may be called a primary transcript when it is a complete complementary copy of a DNA sequence. In some embodiments, an RNA transcript may be called mature RNA when it is, for example, an RNA sequence derived from post-transcriptional processing of a primary transcript.

[0030] In some embodiments, “messenger RNA” or “mRNA” may refer to RNA that does not contain introns and can be translated into proteins by a cell.

[0031] In some embodiments, “sense” RNA may refer to an RNA transcript containing mRNA. In some embodiments, sense RNA may be translated into a protein intracellularly or in vitro.

[0032] In some embodiments, “antisense RNA” may refer to an RNA transcript that may be complementary to all or part of the target RNA (e.g., primary transcript or mRNA). In some embodiments, antisense RNA can be used to block the expression of a target gene. In some embodiments, the complementarity of the antisense RNA may be to any part of a particular gene transcript, i.e., a 5' non-coding sequence, a 3' non-coding sequence, an intron, or a coding sequence. In some embodiments, “functional RNA” may refer to antisense RNA, ribozyme RNA, or other RNA that cannot be translated but may still exert an effect on cellular processes. In some embodiments, the terms “complement” and “reverse complement” may be used interchangeably herein, for example, with respect to mRNA transcripts, and may be used to define antisense RNA for a message.

[0033] In some embodiments, "cDNA" may refer to DNA that is complementary to an mRNA template and can be synthesized from an mRNA template using reverse transcriptase. In some embodiments, the cDNA may be single-stranded or converted to a double-stranded form using a Klenow fragment of DNA polymerase I.

[0034] In some embodiments, “coding region” may refer to a portion of messenger RNA (or a corresponding portion of another nucleic acid molecule, such as a DNA molecule) that can code for a protein or polypeptide. In some embodiments, “non-coding region” may refer to a portion of messenger RNA or other nucleic acid molecule that is not a coding region, including but not limited to, the promoter region, the 5' untranslated region ("UTR"), the 3'UTR, introns, and terminators. In some embodiments, the terms “coding region” and “coding sequence” may be used interchangeably herein. In some embodiments, the terms “non-coding region” and “non-coding sequence” may be used interchangeably herein.

[0035] In some embodiments, "code array" can be abbreviated as "CDS". In some embodiments, "open reading frame" can be abbreviated as "ORF".

[0036] In some embodiments, “gene” may refer to a nucleic acid fragment capable of expressing a functional molecule, such as a specific protein, which may include, but is not limited to, introns, exons, regulatory sequences preceding a coding sequence (5' non-coding sequence), and regulatory sequences following it (3' non-coding sequence). In some embodiments, “native gene” may refer to a naturally occurring gene, for example, one that has its own regulatory sequence.

[0037] In some embodiments, a “mutant gene” may be a gene that has been modified, for example, by human intervention, compared to the corresponding naturally occurring gene. In some embodiments, such a “mutant gene” may have a sequence different from the sequence of the corresponding non-mutant gene by at least one nucleotide addition, deletion, or substitution. In some embodiments, a mutant gene may include changes resulting from a polynucleotide-derived polypeptide system disclosed herein. In some embodiments, a mutant organism may be an organism containing a mutant gene, for example, a mutant plant having an organelle genome containing a mutant gene. In some embodiments, the terms “mutant gene” and “mutant gene” may be used interchangeably herein.

[0038] In some embodiments, the term “SDN” may refer to “site-directed nuclease.” In some embodiments, SDN-induced mutations may include the induction of site-directed, random mutations, the induction of mutations in a given sequence of a particular gene, the substitution or insertion of an entire gene, or any combination thereof. In some embodiments, SDN-induced mutations may be referred to as SDN-1, SDN-2, and SDN-3, respectively.

[0039] In some embodiments, a “codon-modifying gene,” “codon-preferential gene,” or “codon-optimizing gene” may be a gene having a codon usage frequency designed to mimic the preferred codon usage frequency of a host cell in a compartment of interest. In some embodiments, the compartment of interest may include the nucleus, mitochondria, chloroplasts, or any combination thereof.

[0040] In some embodiments, “mature” protein may refer to a polypeptide that has been processed post-translation, for example, one from which any pre- or pro-peptides present in the primary translation product have been removed.

[0041] In some embodiments, the “precursor” protein may refer to the primary product of mRNA translation, e.g., a pre- or pro-peptide, which still exists. In some embodiments, the pre- or pro-peptide may include, for example, an intracellular localization signal.

[0042] In some embodiments, “isolated” can refer to substances such as nucleic acid molecules, proteins, and cells that substantially do not contain, or can otherwise remove from, components that are typically associated with or interact with, a substance in its naturally occurring environment. In some embodiments, isolated polynucleotides can be purified from host cells in which they can naturally occur. In some embodiments, isolated polynucleotides can be obtained using nucleic acid purification methods. In some embodiments, isolated polynucleotides may include, for example, recombinant polynucleotides and chemically synthesized polynucleotides.

[0043] In some embodiments, “heterogeneous” may mean, for example, with respect to the sequence, a sequence that originates from an exotic species or, if it originates from the same species, a sequence that is substantially modified from its natural form in the composition and / or genomic locus by intentional human intervention. In some embodiments, the terms “heterogeneous nucleotide sequence,” “heterogeneous sequence,” “heterogeneous nucleic acid fragment,” and “heterogeneous nucleic acid sequence” may be used interchangeably herein. In some embodiments, “heterogeneous” may mean a nucleic acid sequence that does not naturally exist in the genome. In some embodiments, “heterogeneous” may mean a nucleic acid sequence that has been artificially introduced into the genome. In some embodiments, “heterogeneous” may mean a nucleic acid sequence that is present in the genome as a result of gene editing. In some embodiments, heterogeneous nucleic acids may be exogenous or endogenous to the cell. In some embodiments, “heterogeneous” may mean a nucleic acid sequence that does not naturally exist in one organelle of a cell but is present in another organelle of the same cell. In some embodiments, “heterogeneous” may mean a nucleic acid sequence that has been introduced into the mitochondria of a cell and does not naturally exist in the mitochondria of that cell but is present in the nucleus of that cell. In some embodiments, the methods disclosed herein can produce organelles containing heterologous nucleic acid sequences that are not integrated into the organelle genome. In some embodiments, the heterologous nucleic acid sequences that are not integrated into the organelle genome may include sequences that are part of a plasmid.

[0044] In some embodiments, “recombinant” may refer to an artificial combination of two or more differently separated segments of a sequence, for example, by chemical synthesis or by manipulating isolated segments of nucleic acids using genetic engineering techniques. In some embodiments, “recombinant” may also include references to cells or vectors modified by the introduction of heterologous nucleic acids, or cells derived from such modified cells.

[0045] In some embodiments, “recombinant DNA construct” may refer to a combination of nucleic acid fragments that may not normally be found together in nature. In some embodiments, a recombinant DNA construct may include, for example, regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source. In some embodiments, the sequences in a recombinant DNA construct may be arranged differently from those normally found in nature. In some embodiments, the terms “recombinant DNA construct,” “recombinant DNA molecule,” “recombinant construct,” “DNA construct,” and “construct” may be used interchangeably herein. In some embodiments, a recombinant DNA construct may, in non-limiting examples, be single-stranded, double-stranded, or both single-stranded and double-stranded, linear or circular, DNA, RNA, or a combination of DNA and RNA, plasmid DNA, viral DNA, viral RNA, or viral RNA.

[0046] In some embodiments, “expression” may refer to the production of a functional product. For example, the expression of a nucleic acid fragment may refer to the transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA), and / or the translation of mRNA into a precursor or mature protein.

[0047] In some embodiments, “expression cassette” may refer to a construct containing, for example, polynucleotides, regulatory elements, and polynucleotides that enable the expression of polynucleotides in a host. In some embodiments, the terms “expression cassette” and “expression construct” may be used interchangeably herein.

[0048] In some embodiments, the terms “entry clone” and “entry vector” may be used interchangeably herein.

[0049] In some embodiments, “regulatory sequence” may refer to a nucleotide sequence located, for example, upstream (e.g., 5' non-coding sequence), internal (e.g., within an intron), or downstream (e.g., 3' non-coding sequence) of a coding sequence. In some embodiments, the regulatory sequence may, for example, affect the transcription, RNA processing or stability, or translation of the associated coding sequence. In some embodiments, the regulatory sequence may include, but is not limited to, promoters, translation leader sequences, 5' non-coding sequences, 3' non-coding sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures. In some embodiments, the regulatory sequence may function in “cis” or “trans” configuration. In some embodiments, the nucleic acid molecule regulated by the regulatory sequence does not necessarily have to encode a functional peptide or polypeptide; for example, the regulatory sequence may regulate the expression of short interfering RNA or antisense RNA. In some embodiments, the terms “regulatory sequence” and “regulatory element” may be used interchangeably herein.

[0050] In some embodiments, “promoter” may refer to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments, the promoter may include a core promoter (also known as a minimal promoter) sequence. In some embodiments, the core promoter may be a minimal sequence for direct transcription initiation. In some embodiments, the core promoter may optionally include enhancers or other regulatory elements. In some embodiments, the promoter may be derived entirely from a native gene, composed of different elements derived from different naturally occurring promoters, or further include a synthetic DNA segment. Different promoters may induce gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions.

[0051] In some embodiments, a “functional promoter in a plant” may be a promoter capable of regulating transcription within a plant cell. In some embodiments, the promoter may originate from any suitable source, which may include plant cells and non-plant cells.

[0052] In some embodiments, “tissue-specific promoter” and “tissue-preferred promoter” can be used interchangeably and may refer to a promoter that is primarily expressed in one tissue, one organ, or one cell type. In some embodiments, a tissue-specific promoter may not necessarily be exclusive to one tissue, one organ, or one cell type. In some embodiments, root-preferential promoters may include, for example, the soybean root-specific glutamine synthetase gene, cytosolic glutamine synthetase (GS), root-specific regulatory elements in the GRP1.8 gene of Japanese yam, the root-specific promoter of Agrobacterium (A. tumefaciens) mannopin synthase (MAS), root-specific promoters isolated from Parasponia andersonii and Trema tomentosa, the root-inducing genes of Agrobacterium rhizogenes rolC and rolD, the Agrobacterium wound-inducing TR1' and TR2' genes, the VfENOD-GRP3 gene promoter, and the rolB promoter. In some embodiments, seed-preferential promoters may include seed-specific promoters active during seed development, germination promoters active during seed germination, or any combination thereof. In some embodiments, seed-preferred promoters may include Cim1 (cytokinin-inducing message), cZ19B1 (maize 19kDa zein), milps (myo-inositol-1-phosphate synthase), END1, and END2, or any combination thereof. In some embodiments, for dicotyledonous plants, seed-preferred promoters may include legume β-phaseolin, napin, β-conglycinin, soybean lectin, cluciferin, or any combination thereof. In some embodiments, for monocotyledonous plants, seed-preferred promoters may include maize 15kDa zein, 22kDa zein, 27kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, nud, Zea mays-Rootmet 2 promoter, or any combination thereof.In some embodiments, leaf-preferential promoters may include plant rbcS promoters such as soybean rbcS promoters and maize rbcS promoters, G-maize PEPC1 promoters, or any combination thereof.

[0053] In some embodiments, “ecologically regulated promoter” may refer to a promoter having activity that can be determined by the ecological event.

[0054] In some embodiments, “inducible promoter” may refer to a promoter that selectively expresses a manipulably linked DNA sequence in response to the presence of endogenous or exogenous stimuli, such as chemical compounds (e.g., chemical inducers), or in response to environmental, hormonal, chemical, and / or developmental signals. In some embodiments, inducible or moduloactive promoters may include promoters regulated by light, heat, stress, flood, or drought, plant hormones, wounds, or chemicals, such as ethanol, jasmonate, salicylic acid, or pharmacotoxicity reducers. In some embodiments, pathogen-inducible promoters that may be induced after pathogen infection may include those that regulate the expression of PR proteins, SAR proteins, β-1,3-glucanase, chitinase, or any combination thereof. In some embodiments, stress-inducible promoters may include plant RAB17 promoters, such as the maize RAB17 promoter. In some embodiments, chemically inducible promoters include the maize ln2-2 promoter, the maize GST promoter, the tobacco PR-1a promoter, or any combination thereof. In some embodiments, the maize ln2-2 promoter can be activated by a benzenesulfonamide herbicide phytotoxicity reducer. In some embodiments, the maize GST promoter can be activated by a hydrophobic electrophile. In some embodiments, the maize GST promoter can be used as a pre-germination herbicide. In some embodiments, the tobacco PR-1a promoter can be activated by salicylic acid. In some embodiments, chemically modulated promoters include steroid-responsive promoters, such as glucocorticoid-inducible promoters, tetracycline-inducible and tetracycline-inhibitory promoters.

[0055] In some embodiments, “constitutive promoter” may refer to a promoter that is active in all or most tissues or cell types of an organism at all or most developmental stages. In some embodiments, in a promoter classified as “constitutive” (e.g., ubiquitin), some variation in the absolute level of expression may exist in different tissues or stages. In some embodiments, the terms “constitutive promoter” and “tissue-independent promoter” may be used interchangeably herein. In some embodiments, constitutive promoters include plant actin promoters such as the Rsyn7 promoter core promoter, the core CaMV 35S promoter, the rice actin promoter and the maize actin promoter; plant ubiquitin promoters such as the maize ubiquitin promoter and the soybean ubiquitin promoter; plant GOS2 promoters such as the pEMU, MAS promoter, ALS promoter and the maize GOS2 promoter; plant U6 polymerase III promoters such as the soybean GM-EF1 A2 promoter, the maize U6 polymerase III promoter and the soybean U6 polymerase III promoter (GM-U6-9.1 and GM-U6-13.1); and any combination thereof.

[0056] In some embodiments, the enhancer element may be any nucleic acid molecule that, when functionally linked to the promoter regardless of its relative position, increases the transcription of the nucleic acid molecule. In some embodiments, the enhancer may be an innate element of the promoter, or a heterogeneous element inserted to improve the level or tissue specificity of the promoter.

[0057] In some embodiments, a repressor (which may also be referred to herein as a silencer) can be defined as any nucleic acid molecule that, when functionally linked to a promoter regardless of its relative position, inhibits transcription.

[0058] In some embodiments, the “translation leader sequence” may refer to a polynucleotide sequence located between the promoter sequence and the coding sequence of a gene. In some embodiments, the translation leader sequence may be present in fully processed mRNA upstream of the translation initiation sequence. In some embodiments, the translation leader sequence may influence the processing of the primary transcript to the mRNA, mRNA stability, or translation efficiency.

[0059] In some embodiments, “transcription terminator,” “terminator sequence,” or “terminator” may refer to a DNA sequence that, when manipulably ligated to the 3' end of a polynucleotide sequence to be expressed, can terminate transcription from the polynucleotide sequence. In some embodiments, transcription termination may refer to a process that can stop RNA synthesis by RNA polymerase, releasing both RNA and the enzyme from the DNA template.

[0060] In some embodiments, “operably linked” may refer to the association of fragments in a single fragment (e.g., a polynucleotide or polypeptide) or in a single complex such that the function of one can be modulated by the function of the other. In some embodiments, linkage may be covalent or non-covalent. In some embodiments, with respect to a nucleic acid fragment, a promoter can be operably linked to a nucleic acid fragment if the promoter can modulate the transcription of the nucleic acid fragment. In some embodiments, with respect to a polypeptide, an organelle-targeting peptide can be operably linked to a polypeptide if the organelle-targeting peptide can transport the polypeptide to the relevant organelle. In some embodiments, with respect to a complex, a guide RNA / Cas polypeptide complex can be operably linked to a Cas polypeptide if the guide RNA can cleave a target sequence induced by the guide RNA.

[0061] In some embodiments, “phenotype” may refer to detectable characteristics of a cell or organism.

[0062] In some embodiments, the term “introduced” may mean providing a polynucleic acid (e.g., an expression construct) or protein to a cell. In some embodiments, “introduced” may include references to incorporating a nucleic acid into a eukaryotic or prokaryotic cell, for example, so that the nucleic acid can be incorporated into the cell’s genome. In some embodiments, “introduced” may include references to transiently providing a nucleic acid or protein to a cell. In some embodiments, “introduced” may include references to stable or transient gene editing methods. In some embodiments, “introduced” may include references to stable or transient transformation methods. Introduction may include sexual mating. In some embodiments, “introduced” may include “transfection,” “transformation,” or “transduction,” for example, inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell. In some embodiments, “introduced” may include a reference to incorporating a nucleic acid fragment into a eukaryotic or prokaryotic cell, in which case the nucleic acid fragment may be incorporated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0063] In some embodiments, the “edited mitochondrial genome” may include (i) the substitution of at least one nucleotide, (ii) the deletion of at least one nucleotide, (iii) the insertion of at least one nucleotide, or (iv) the introduction of any combination of (i) to (iii). In some embodiments, substitution and replacement are used herein to mean the exchange of an existing nucleotide for an alternative nucleotide. In some embodiments, a cell may include an edited mitochondrial genome having at least one nucleotide substitution, deletion, or insertion. In some embodiments, a cell may include transformed mitochondria, and transformed mitochondria include an edited mitochondrial genome.

[0064] In some embodiments, “transformed cells” can be any cells into which nucleic acid fragments (e.g., recombinant DNA constructs) have been introduced or edited.

[0065] In some embodiments, “transformation” as used herein may refer to a stable transformation. In some embodiments, transformation may refer to a transient transformation.

[0066] In some embodiments, “stable transformation” may refer to the introduction of a nucleic acid fragment into the genome of a host organism (e.g., that of the nucleus, mitochondria, or plastids) resulting in genetically stable inheritance. In some embodiments, once stably transformed, the nucleic acid fragment can be stably incorporated into the genome of the host organism and all subsequent generations.

[0067] In some embodiments, “transient transformation” may refer to introducing nucleic acid fragments into the nucleus or DNA-containing cytoplasmic organelles (e.g., mitochondria, plastids) to edit or modify the host organism’s nucleus or organelle genome, thereby resulting in gene expression that does not involve genetically stable inheritance.

[0068] In some embodiments, a host organism containing a transformed nucleic acid fragment may be referred to as a “transgenic” organism.

[0069] In some embodiments, “transformation cassette” may refer to a construct having elements that promote the transformation of specific host cells. In some embodiments, the terms “transformation cassette” and “transformation construct” may be used interchangeably herein.

[0070] In some embodiments, "homoplasmic," when used in relation to mitochondria, may refer to eukaryotic cells in which all copies of mitochondrial DNA are identical. In some embodiments, "heteroplasmic" may refer to eukaryotic cells in which all copies of mitochondrial DNA are not identical.

[0071] In some embodiments, when used in reference to plastids, “homoplasmic” may refer to eukaryotic cells in which all copies of plastid DNA are identical. In some embodiments, “heteroplasmic” may refer to eukaryotic cells in which all copies of plastid DNA are not identical.

[0072] In some embodiments, an "allele" may be one of several alternative forms of a gene occupying a given locus on a chromosome. In some embodiments, a diploid plant may be homozygous at a given locus if the alleles present at that locus on a pair of homologous chromosomes are the same. In some embodiments, a diploid plant may be heterozygous at a given locus on a pair of homologous chromosomes if the alleles present at that locus are different. In some embodiments, a plant may be hemizygous at a locus if the transgene is present on one of a pair of homologous chromosomes in a diploid plant.

[0073] In some embodiments, “organelle” may be a DNA-containing organelle of a cell. In some embodiments, the organelle may be the nucleus, mitochondria, plastids (e.g., chloroplasts), or any combination thereof. In some embodiments, the organelle may be a DNA-containing cytoplasmic organelle. In some embodiments, the organelle may be mitochondria, plastids (e.g., chloroplasts), or any combination thereof. In some embodiments, the plastid may be proplastids, etioplasts, leucoplasts, amyloplasts, elaioplasts, protinoplasts, chromoplasts, chloroplasts, geronoplasts, or any combination thereof.

[0074] In some embodiments, the terms “organelle-specific” and “organelle-preferential” can be used interchangeably, and when used to describe a regulatory element (e.g., an organelle-specific promoter), it refers to a regulatory element that is functional within a given cell (e.g., a plant cell) but is not necessarily exclusive within an organelle (e.g., mitochondria, plastids).

[0075] In some embodiments, the organelle-specific regulatory domain may be derived from the organelle polynucleotide of interest (e.g., mitochondrial polynucleotide, plastidopolynucleotide). In some embodiments, the organelle-specific regulatory domain may comprise all or part of the nucleic acid sequence of the organelle polynucleotide of interest. In some embodiments, the organelle-specific regulatory domain may be 100% or less identical to all or part of the organelle polynucleotide of interest (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical).

[0076] In some embodiments, the terms “mitochondrial-specific” and “mitochondrial-preferential” can be used interchangeably, and when used to describe a regulatory element (e.g., a mitochondrial-specific promoter), it refers to a regulatory element that functions within a given cell (e.g., a plant cell) that is dominant in mitochondria but not necessarily exclusive.

[0077] In some embodiments, the terms “plastido-specific” and “plastido-preferential” can be used interchangeably and, when used to describe a regulatory element (e.g., a plastid-specific promoter), refer to a regulatory element that functions within a given cell (e.g., a plant cell) that is dominant in plastids but not necessarily exclusive to them.

[0078] In some embodiments, the terms “chloroplast-specific” and “chloroplast-preferential” can be used interchangeably, and when used to describe a regulatory element (e.g., a chloroplast-specific promoter), it refers to a regulatory element that functions within a given cell (e.g., a plant cell) that is dominant in chloroplasts but not necessarily exclusive.

[0079] In some embodiments, the terms “mitochondrial genome” and “genomics of mitochondria” can be used interchangeably and refer to nucleic acid sequences present within endogenous mitochondrial genetic elements. In some embodiments, the mitochondrial genome may be edited by appending sequences (e.g., heterologous sequences) to endogenous mitochondrial genetic elements. In some embodiments, self-replicating heterologous episomal elements (e.g., plasmid DNA) introduced into mitochondria are considered independent genetic elements but are not considered part of the mitochondrial genome.

[0080] In some embodiments, the terms “plastid genome,” “chloroplast genome,” “plastid genome,” and “chloroplast genome” can be used interchangeably and refer to nucleic acid sequences present within endogenous plastid genetic elements. In some embodiments, the plastid genome may be edited by appending sequences (e.g., heterologous sequences) to endogenous plastid genetic elements. In some embodiments, self-replicating heterologous episomal elements (e.g., plasmid DNA) introduced into a plastid are considered independent genetic elements but are not considered part of the plastid genome.

[0081] In some embodiments, the “chloroplast transit peptide” may be an amino acid sequence capable of directing a protein to a chloroplast or other plastid form present within the cell. In some embodiments, the chloroplast transit peptide may be translated in conjunction with an intracellular protein in which a protein can be produced. In some embodiments, the terms “chloroplast transit peptide,” “plastid transit peptide,” “chloroplast-targeting peptide,” and “plastid-targeting peptide” may be used interchangeably herein. The “chloroplast transit sequence” may refer to a nucleotide sequence capable of encoding a chloroplast transit peptide.

[0082] In some embodiments, the “signal peptide” may be an amino acid sequence that can guide the protein into the secretory system. The signal peptide can be translated together with the protein. For example, if the protein is guided into the vacuole, a vacuolar targeting signal (described above) may be added, or if it is guided into the endoplasmic reticulum, an endoplasmic reticulum retention signal (described above) may be added. If the protein is guided into the nucleus, all existing signal peptides may be removed, and a nuclear localization signal may be included.

[0083] In some embodiments, the “mitochondrial-targeting peptide” may be an amino acid sequence capable of leading a precursor protein to mitochondria. In some embodiments, the terms “mitochondrial-targeting peptide,” “mitochondrial signal peptide,” and “mitochondrial transit peptide” may be used interchangeably herein.

[0084] In some embodiments, “organelle-targeted polynucleotide” may be a nucleotide sequence that can lead to the translocation of a polynucleotide into an organelle. In some embodiments, the terms “organelle-targeted polynucleotide,” “organelle-targeted nucleic acid,” and “organelle-targeted nucleic acid sequence” may be used interchangeably herein. In some embodiments, the organelle-targeted polynucleotide may be directed, for example, to a plastid (“plastiide-targeted polynucleotide”) or a mitochondria (“mitochondrial-targeted polynucleotide”). In some embodiments, the polynucleotide may be RNA (“organelle-targeted RNA”), DNA (“organelle-targeted DNA”), or a combination of RNA and DNA. In some embodiments, organelle-targeted RNA directed to a plastid may be referred to as “plastiide-targeted RNA”. In some embodiments, the terms “plastiide-targeted RNA,” “chloroplast-targeted RNA,” and “transit RNA” may be used interchangeably herein. In some embodiments, organelle-targeted RNA directed to a mitochondria may be referred to as “mitochondrial-targeted RNA”.

[0085] In some embodiments, RNA can be transferred into mitochondria. In some embodiments, one such mitochondrial-targeting RNA may be yeast tRNALys. In some embodiments, yeast tRNALys and its variants can be transferred into human mitochondria. In some embodiments, another RNA that can be transferred into mitochondria may be 5S rRNA. In some embodiments, 5S rRNA may function as a vector for delivering heterologous RNA sequences to, for example, mitochondria (e.g., human). In some embodiments, RNA can be used with the compositions and methods of the present disclosure to target organelles (e.g., mitochondria).

[0086] In some embodiments, RNA can be transferred into a plastid. In some embodiments, plastid-targeting RNAs that can mediate the transfer of bound heterologous RNA include vd-5'UTR (e.g., a virus-like ncRNA sequence acting as a 5'UTR) and eIF4E1 mRNA. In some embodiments, RNA can be used in conjunction with the compositions and methods of this disclosure to target an organelle (e.g., a plastid).

[0087] In some embodiments, as used herein, “fusion” may refer to a protein and / or nucleic acid comprising one or more non-natural sequences (e.g., a portion). In some embodiments, any of the molecules described herein (e.g., nucleic acids, proteins, polypeptides, polynucleic acids, Cas proteins, guide polynucleotides) may be operated as a fusion. In some embodiments, a fusion may comprise one or more identical non-natural sequences. In some embodiments, a fusion may comprise one or more different non-natural sequences. In some embodiments, a fusion may be a chimera. In some embodiments, a fusion may comprise a nucleic acid affinity tag. In some embodiments, a fusion may comprise a barcode. In some embodiments, a fusion may comprise a peptide affinity tag. In some embodiments, a fusion may result in the intracellular localization of a site-specific polypeptide. In some embodiments, a fusion may provide a non-natural sequence (e.g., an affinity tag) that can be used for tracking or purification. In some embodiments, a fusion may be a small molecule such as biotin, or a dye such as Alexafluor dye, cyanine 3 dye, cyanine 5 dye, or any combination thereof.

[0088] In some embodiments, the fusion can refer to any protein having a functional effect. In some embodiments, the fusion protein may have deaminase activity, cytidine deaminase activity (U.S. Patent Application Publication 20150166980, incorporated herein by reference), adenine deaminase activity (U.S. Patent Application Publication 20180073012, incorporated herein by reference), uracil glycosylase inhibitor activity (U.S. Patent Application Publication 20170121693, incorporated herein by reference), methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombination activity The effects may include lyase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. In some embodiments, the effector protein can modify a genomic locus. In some embodiments, the fusion protein may be a fusion in the Cas protein. In some embodiments, the Cas protein may be a modified form that has nickase activity or has substantial nucleic acid cleavage activity. In some embodiments, the fusion protein may be a non-natural sequence within the Cas protein.

[0089] In some embodiments, “silencing,” as used herein in relation to a target gene, may refer to the suppression of the level of mRNA or protein / enzyme expressed by the target gene, and / or the level of enzyme activity or protein functionality. In some embodiments, the terms “suppression,” “suppress,” and “silencing” are interchangeable herein and may include reducing, decreasing, degrading, reducing, inhibiting, eliminating, or preventing. In some embodiments, “silencing” or “gene silencing” may occur by any suitable mechanism. In some embodiments, non-limiting examples of silencing include antisense, co-suppression, viral suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, small RNA-based approaches, and any combination thereof.

[0090] In some embodiments, repression of gene expression can also be achieved by the use of, for example, artificial miRNA precursors, ribozyme constructs, and gene disruption. In some embodiments, modified plant miRNA precursors may be used, which are modified, for example, to replace the miRNA coding region with a sequence designed to produce a miRNA that leads to a desired nucleotide sequence. In some embodiments, gene disruption can be achieved by the use of transpositionable elements or by the use of chemicals that induce site-directed mutations.

[0091] Sequence identity, similarity, and variation In some embodiments, the calculation of sequence alignment and percentages of identity or similarity may be determined using various comparison methods designed to detect homologous sequences, including but not limited to the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, WI). In some embodiments, when sequence analysis software is used for the analysis, the analysis results may be based on the “default values” of the program mentioned. In some embodiments, as used herein, “default values” may mean any set of values ​​or parameters that are initially loaded by the software when it is first initialized.

[0092] In some embodiments, the “Clustal V alignment method” is labeled as Clustal V and may correspond to the alignment method found, for example, in the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, WI). In some embodiments, for multiple alignments, the default values ​​may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. In some embodiments, the default parameters for pairwise alignment and identity percentage calculation for protein sequences using the Clustal method may be, for example, KTUPLE=1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5. In some embodiments, these parameters for nucleic acids may be, for example, KTUPLE=2, GAP PENALTY=5, WINDOW=4, and DIAGONALS SAVED=4. In some embodiments, after aligning sequences using the Clustal V program, the "identity percentage" and "deviation" values ​​can be obtained by checking the "sequence distance" table in the same program.

[0093] In some embodiments, the "Clustal W alignment method" is labeled as Clustal W and may correspond to the alignment method found, for example, in the MEGALIGN® v6.1 program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, WI). In some embodiments, the default parameters for multiple alignments may correspond to, for example, GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergence Sequences=30%, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, and DNA Weight Matrix=IUB. In some embodiments, after aligning sequences using the Clustal W program, the "identity percentage" value can be obtained by checking the "sequence distance" table in the same program.

[0094] In some embodiments, sequence identity / similarity values ​​can also be obtained using GAP version 10 (GCG, ACCELRYS®, San Diego, CA), which uses, for example, the following parameters: a gap generation penalty weight of 50 and a gap length extension penalty weight of 3, as well as identity % and similarity % for nucleotide sequences using the nwsgapdna.cmp scoring matrix, a GAP creation penalty weight of 8 and a gap length extension penalty of 2, as well as identity % and similarity % for amino acid sequences using the BLOSUM62 scoring matrix. In some embodiments, GAP can use an algorithm to find alignments of two complete sequences that can maximize the number of matches and minimize the number of gaps. In some embodiments, GAP can consider all possible alignments and gap locations. In some embodiments, GAP can create alignments with the maximum number of matched bases and the minimum number of gaps, for example, by using gap generation penalties and gap length extension penalties in units of matched bases.

[0095] In some embodiments, "BLAST" may be a search algorithm provided by the National Center for Biotechnology Information (NCBI) that can be used to find similarity regions between biological sequences. In some embodiments, BLAST can compare nucleotide or protein sequences with a sequence database. In some embodiments, BLAST can calculate the statistical significance of the match to identify sequences that are sufficiently similar to the query sequence so that the similarity is not predicted to be random. In some embodiments, BLAST can report the identified sequences and their local alignments with respect to the query sequence.

[0096] In some embodiments, the terms “conserved domain” or “motif” may refer to a set of amino acids conserved at specific locations along the aligned sequence of evolutionarily related proteins. In some embodiments, amino acids at other locations may vary among homologous proteins, but amino acids highly conserved at specific locations may represent, for example, amino acids essential to the structure, stability, or activity of the protein.

[0097] In some embodiments, conserved domains or motifs can be identified by being highly conserved in the aligned sequences of a family of protein homologs. In some embodiments, conserved domains can be used as identifiers or "signatures" to determine, for example, whether a protein with a newly determined sequence belongs to a previously identified protein family.

[0098] In some embodiments, polynucleotide and polypeptide sequences, their variants, and the structural relationships of these sequences can be described by the terms “homologous,” “homonymous,” “substantially identical,” “substantially similar,” and “substantially corresponding,” which are used interchangeably herein. In some embodiments, these may refer to polypeptides or nucleic acid fragments in which a change in one or more amino acids or nucleotide bases does not affect the molecular function, such as the ability to mediate gene expression or produce a particular phenotype. In some embodiments, these terms may also refer to modifications of a nucleic acid fragment in which the functional properties of the resulting nucleic acid fragment are not substantially altered compared to the original unmodified fragment. In some embodiments, these modifications may include deletions, substitutions, insertions, or any combination thereof of one or more nucleotides in the nucleic acid fragment.

[0099] In some embodiments, substantially similar nucleic acid sequences may be defined by their ability to hybridize with any portion of the sequences exemplified herein or the nucleotide sequences disclosed herein (e.g., under moderately strict conditions, e.g., 0.5X SSC, 0.1% SDS, 60°C). In some embodiments, substantially similar nucleic acid sequences may be functionally equivalent to any of the nucleic acid sequences disclosed herein. In some embodiments, the strictness conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distant related organisms, to highly similar fragments, such as genes replicating functional enzymes from nearby related organisms. In some embodiments, the strictness conditions can be determined by washing after hybridization.

[0100] In some embodiments, the term “selectively hybridize” may refer to hybridizing a nucleic acid sequence to a specific nucleic acid target sequence to a detectably greater extent (e.g., at least twice the background) than hybridization to a non-target nucleic acid sequence and substantial exclusion of the non-target nucleic acid, for example, under strict hybridization conditions. In some embodiments, the selectively hybridizing sequences may have, for example, at least about 80% sequence identity, or 90% sequence identity, or up to 100% sequence identity (i.e., fully complementary) to one another.

[0101] In some embodiments, the terms “strict conditions” or “strict hybridization conditions” may refer to conditions under which a probe can selectively hybridize to its target sequence in an in vitro hybridization assay. In some embodiments, strict conditions may be sequence-dependent. In some embodiments, strict conditions may vary in different situations. In some embodiments, target sequences that are 100% complementary to the probe can be identified by controlling the strictness of hybridization and / or washing conditions (homologous probing).

[0102] In some embodiments, the strictness requirement can be adjusted to allow for some mismatches within the sequences so that a lower degree of similarity can be detected (heterogeneous probing). In some embodiments, the probe may be less than approximately 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

[0103] In some embodiments, strict conditions may include conditions where the salt concentration is less than about 1.5 M of Na ions. In some embodiments, strict conditions may include conditions where the salt concentration is less than about 0.01 to 1.0 M of Na ions (or other salts) at pH 7.0 to 8.3. In some embodiments, strict conditions may include a temperature of about 30°C with a short probe (e.g., 10 to 50 nucleotides). In some embodiments, strict conditions may include a temperature of at least about 60°C with a long probe (e.g., more than 50 nucleotides). In some embodiments, strict conditions can also be achieved by adding an stabilizer such as formamide. In some embodiments, exemplary low-strictness conditions may include, for example, hybridization at 37°C with a buffer solution of 30 to 35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), and washing at 50 to 55°C with 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate). In some embodiments, exemplary moderate-strictness conditions may include hybridization at 37°C with 40-45% formamide, 1M NaCl, and 1% SDS, and washing at 55-60°C with 0.5X-1X SSC. In some embodiments, exemplary high-strictness conditions may include, for example, hybridization at 37°C with 50% formamide, 1M NaCl, and 1% SDS, and washing at 60-65°C with 0.1X SSC.

[0104] In some embodiments, “sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences may refer to nucleic acid bases or amino acid residues in two sequences that are identical when aligned to correspond to a particular comparison window.

[0105] In some embodiments, the term “percentage of sequence identity” may refer to a value determined by comparing two optimally aligned sequences across a comparison window. In some embodiments, portions of the polynucleotide or polypeptide sequences in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which may or may not include additions or deletions) for optimal alignment of the two sequences. In some embodiments, the percentage can be calculated, for example, by determining the number of positions in which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In some embodiments, the percentage of sequence identity may include, but is not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%. In some embodiments, array identity may include an integer percentage from 50% to 100%. In some embodiments, these identities can be determined using one of the programs described herein.

[0106] In some embodiments, sequence identity may be useful in identifying polypeptides derived from other species or naturally or synthetically modified polypeptides having the same or similar function or activity. In some embodiments, the percentage may include, but is not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, sequence identity (e.g., amino acid sequence identity) may include integer percentages from 50% to 100%. In some embodiments, sequence (e.g., amino acid) identity may include, for example, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0107] Definitions, traits, and processes related to plants In some embodiments, “plant” may include references to the whole plant, plant organs, plant tissues, plant reproductive bodies, seeds, and plant cells, as well as their offspring. In some embodiments, plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

[0108] In some embodiments, “bulbils” may include meiotic and / or mitotic products capable of propagating new plants. In some embodiments, the reproductive body may include plant seeds, spores, and parts that can function as a means of vegetative propagation, such as corms, tubers, lateral branches, or stolons. In some embodiments, the reproductive body may include grafts, which can produce living organisms by grafting a part of one plant onto another part of a different plant (one of yet another different species). In some embodiments, the reproductive body may include plants and seeds produced by cloning, or by collecting meiotic products, or by collecting meiotic products and enabling them to form embryos or fertilized eggs (naturally or with human intervention).

[0109] In some embodiments, “offspring” may include all subsequent generations of the plant.

[0110] In some embodiments, the terms “monocot” and “monocotyledonous plant” can be used interchangeably herein. In some embodiments, monocotyledonous plants may include grasses.

[0111] In some embodiments, the terms “dicot” and “dicotyledonous plant” may be used interchangeably herein. In some embodiments, dicotyledonous plants may include, for example, the families Brassicaceae, Leguminosae, and Solanaceae.

[0112] In some embodiments, “transgenic plant” may refer to a plant whose genome may contain heterologous polynucleotides. In some embodiments, heterologous polynucleotides may be stably incorporated into the genome (e.g., nucleus, plastids, mitochondria) so that the polynucleotides can be passaged to the next generation. In some embodiments, heterologous polynucleotides may be incorporated into the genome alone or as part of a recombinant DNA construct.

[0113] In some embodiments, “transgenic plant” may refer to a plant that may contain one or more heterologous polynucleotides in its genome. In some embodiments, each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0114] In some embodiments, multiple traits can be introduced into a crop plant and may be referred to as a gene stacking approach. In some embodiments, gene stacking can be used, for example, to develop genetically improved germplasm. In some embodiments, multiple genes conferring various desired features can be introduced into a plant. In some embodiments, gene stacking can be achieved by many means, including but not limited to simultaneous transformation, retransformation, and crossing lines with different introduced genes. In some embodiments, as used herein, the term “stacked” may include having multiple traits present in the same plant (e.g., both traits are integrated into the nuclear genome, one trait is integrated into the nuclear genome and the other into the organelle genome, or both traits are integrated into the organelle genome).

[0115] In some embodiments, the terms “crossed,” “cross,” or “crossing” in the context of this disclosure may mean the fusion of gametes (e.g., by pollination) to produce offspring (e.g., cells, seeds, or plants). In some embodiments, the terms may encompass both sexual crossing (e.g., pollination of one plant by another) and self-crossing (e.g., self-pollination (where pollen and eggs originate from the same plant or genetically identical plants)).

[0116] In some embodiments, the term “maternal inheritance” may refer to the transmission of traits that depend solely on the genomic characteristics of the female gamete.

[0117] In some embodiments, the term “patrilineal inheritance” may refer to the transmission of traits that depend solely on the genomic characteristics of the male gamete.

[0118] In some embodiments, the term “gene transfer” may refer to the transfer of a desired allele at a locus from one genetic background to another. In some embodiments, the transfer of a desired allele at a particular locus can be transmitted to at least one offspring plant via sexual mating between two parent plants, where at least one of the parent plants has the desired allele in its genome. In some embodiments, allele transfer may occur by recombination between two donor genomes, for example, in a fusion protoplast, where at least one of the donor protoplasts has the desired allele in its genome. In some embodiments, the desired allele may be, for example, a marker or QTL transgene or a selected allele.

[0119] In some embodiments, a “plant-optimized nucleotide sequence” may be a nucleotide sequence optimized for increased expression in plants, particularly in a given plant or one or more target plants. In some embodiments, a plant-optimized nucleotide sequence can be synthesized by modifying a protein-coding nucleotide sequence using plant-preferred codons to improve expression. In some embodiments, the use of host-preferred codons may be utilized for codon optimization. In some embodiments, the frequency of codon use may be designed to mimic the preferred frequency of codon use in a host cell in a target compartment, e.g., the nucleus, mitochondria, or chloroplasts.

[0120] In some embodiments, plant-preferred genes can be synthesized. In some embodiments, gene expression in a plant host can be enhanced by further sequence modifications. In some embodiments, these may include, for example, the exclusion of one or more sequences encoding spurious polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and sequences that may be detrimental to gene expression. In some embodiments, the GC content of the sequence may be adjusted to a given average level in a plant host, calculated, for example, by reference to the gene expressed in the host plant cell. In some embodiments, where possible, the sequence may be modified to avoid one or more predicted hairpin secondary mRNA structures. In some embodiments, the “plant-optimized nucleotide sequence” of this disclosure may include one or more such sequence modifications.

[0121] In some embodiments, “trait” may refer to, for example, the physiological, morphological, biochemical, or physical characteristics of a plant or specific plant material or cell. In some examples, characteristics may be visually observable to the human eye, such as the size of seeds or plants, or measured by biochemical techniques such as detecting the protein, starch, or oil content of seeds or leaves, or by observing metabolic or physiological processes, such as measuring tolerance to water deficiency or specific salt or sugar concentrations, or by observing gene expression levels, or by agricultural observations such as osmotic stress tolerance or yield.

[0122] In some embodiments, “agricultural characteristics” may be measurable parameters including, but not limited to, abiotic stress tolerance, greenness, yield, growth rate, biomass, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in plant tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in plant tissue, total plant protein content, fruit protein content, seed protein content, protein content in plant tissue, drought tolerance, nitrogen uptake, rooting, harvest index, stem entanglement, plant height, panicle height, panicle length, salt tolerance, early seedling vigor, and seedling emergence under cold stress.

[0123] Plant herbicide resistance In some embodiments, the protein resulting from the expression of a “herbicide-resistant protein” or “herbicide-resistant coding nucleic acid molecule” may include, for example, a protein that can confer to cells the ability to tolerate higher concentrations of herbicides compared to cells that do not express the protein. In some embodiments, the herbicide-resistant protein may have enzymatic activity. In some embodiments, the herbicide-resistant protein may have enzymatic activity in the presence of a herbicide that targets the above enzymatic activity. In some embodiments, the herbicide-resistant protein may have enzymatic activity that results in the degradation and / or inactivation of the herbicide. In some embodiments, the herbicide-resistant protein may be monomeric or polymeric. In some embodiments, the herbicide-resistant protein may comprise a single polypeptide. In some embodiments, the herbicide-resistant protein may comprise two or more different polypeptides. In some embodiments, the terms “herbicide resistance protein,” “herbicide-resistant protein,” “herbicide tolerance protein,” and “herbicide tolerant protein” may be used interchangeably herein when used to describe molecules that can confer the ability of cells (or plants) to tolerate higher concentrations of herbicides compared to cells (or plants) that do not express the molecule (e.g., protein, enzyme, subunit, or nucleic acid).

[0124] In some embodiments, the herbicide-resistant protein, or the protein resulting from the expression of herbicide-resistant coding nucleic acid molecules, may include a protein that can confer to cells the ability to tolerate herbicide concentrations for longer periods than cells that do not express the protein. In some embodiments, the herbicide-resistant trait can be introduced into plants, for example, by a gene encoding herbicide resistance. In some embodiments, genes encoding herbicide resistance include, for example, a gene that acts to transmit resistance to acetolactate synthase (ALS) inhibitors such as sulfonylurea herbicides, a gene that acts to transmit resistance to glutamine synthase inhibitors such as phosphinothricin or basta (e.g., bar gene, pat gene), a gene that acts to transmit resistance to EPSP synthase gene inhibitors such as glyphosate, a gene that acts to transmit resistance to HPPD inhibitors, a gene that acts to transmit resistance to acetyl coenzyme A carboxylase (ACCase) inhibitors, and a gene that acts to transmit resistance to protoporphyrinogen oxidase (PPO or PROTOX) inhibitors.

[0125] In some embodiments, genes useful for conferring herbicide resistance to plants may include genes encoding herbicide resistance proteins. In some embodiments, herbicide resistance proteins may include herbicide resistance forms of acetyl coenzyme A carboxylase (ACCase), 4-hydroxyphenylpyrubate dioxygenase (HPPD), sulfonylurea-resistant acetolactic acid synthase, imidazolinone-resistant acetolactic acid synthase, glyphosate-resistant 5-enolpyruvirschimate-3-phosphate synthase (EPSPS), glyphosate-resistant glyphosate oxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), phosphinotricin acetyltransferase (PAT), protoporphyrinogen oxidase (PPO or PROTOX), auxin enzymes or receptors, P450 polypeptides, or any combination thereof.

[0126] In some embodiments, as used herein, “hydroxyphenylpyrubate dioxygenase” and “HPPD,” “4-hydroxyphenylpyrubate (or pyruvate) dioxygenase (4-HPPD),” and “p-hydroxyphenylpyrubate (or pyruvate) dioxygenase (p-OHPP)” may be synonymous and may refer to a non-heme iron-dependent oxygenase that catalyzes the conversion of 4-hydroxyphenylpyrubate to homogentisate. In some embodiments, in organisms that degrade tyrosine, the reaction catalyzed by HPPD may be a second step in the pathway. In some embodiments, in plants, the formation of homogentisate may be required for the synthesis of plastoquinone and tocopherol, which can function as redox cofactors. In some embodiments, a polynucleotide molecule encoding herbicide-resistant hydroxyphenylpyrubate dioxygenase (HPPD) can confer resistance to HPPD inhibitors.

[0127] In some embodiments, as used herein, “HPPD inhibitor” may include any compound or combination thereof that can reduce the ability of HPPD to catalyze the conversion of 4-hydroxyphenylpyrubate to homogentisate. In certain embodiments, HPPD inhibitors may include herbicidal inhibitors of HPPD. In some embodiments, non-limiting examples of HPPD inhibitors include triketones (such as mesotrione, sulcotrione, topramezone, and tembotrione), isoxazoles (such as pyrasulfolol and isoxaflutol), pyrazoles (such as benzofenap, pyrazoxifen, and pyrazolinates), and benzobicyclon. In some embodiments, agriculturally acceptable salts of various inhibitors may include salts (e.g., cations or anions) for forming salts for agricultural or horticultural use.

[0128] In some embodiments, “herbicide-resistant ALS polypeptide,” “herbicide-tolerant ALS polypeptide,” and “ALS inhibitor-resistant polypeptide” can be used interchangeably and may include any polypeptide that, when expressed in a plant, can confer resistance to at least one acetolactate synthase (ALS) inhibitor. In some embodiments, the ALS inhibitor may include, for example, sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl oxy(thio)benzoate, and / or sulfonylaminocarbonyltriazolinone herbicides. In some embodiments, ALS mutations can be classified into various classes with respect to resistance to, for example, sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate, and sulfonylaminocarbonyltriazolinone. In some embodiments, ALS mutations may include mutations having one or more of the following characteristics: (1) broad resistance to all five groups (i.e., sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate, and sulfonylaminocarbonyltriazolinone); (2) resistance to four of these groups (e.g., sulfonylurea, triazolopyrimidine, pyrimidinyl(thio)benzoate, and sulfonylaminocarbonyltriazolinone); (3) resistance to imidazolinone and pyrimidinyl(thio)benzoate; (4) resistance to sulfonylurea and triazolopyrimidine; and (5) resistance to sulfonylurea and imidazolinone.

[0129] In some embodiments, imidazolinone may include imazapyr, imazapic, imazetapyr, imazamox, imazametabenz, imazakine, any salt of these, any stereoisomer of these, or any combination thereof. In some embodiments, triazolopyrimidine may include penoxyslam, chloranslam-methyl, dicloslam, floraslam, flumethoslam, metoslam, pyroxyslam, any salt of these, any stereoisomer of these, or any combination thereof. In some embodiments, pyrimidinyl benzoate may include bispyribac-sodium, pyribenzoxime, pyrithiobac-sodium, any salt of these, any stereoisomer of these, or any combination thereof. In some embodiments, sulfonanilide may include pyrimisulfan, triafamone, any salt of these, any stereoisomer of these, or any combination thereof. In some embodiments, sulfonylaminocarbonyltriazolinone may include fulcarbazone-sodium, propoxycarbazone-sodium, thiencarbazone-methyl, any salt thereof, any stereoisomer thereof, or any combination thereof.In some embodiments, sulfonylurea is amidesulfuron, azimsulfuron, bensulfuron-methyl, chlorimulon-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, etamethosulfuron-methyl, ethoxysulfuron, fluzasulfuron, flucetosulfuron, flupyrsulfuron-methyl-na, horamsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron-methyl-na, mesosulfuron-methyl, metazosulfuron, metosulfuron-methyl This may include nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron-methyl, propyrisulfuron, prosulfuron, pyrazosulfuron-ethyl, limsulfuron, sulfomethuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenulon-methyl, trifloxysulfuron-na, triflusulfuron-methyl, tritosulfuron, any salt of any of these, any stereoisomer of any of these, or any combination thereof.

[0130] In some embodiments, polynucleotide molecules encoding proteins involved in herbicide resistance may include, for example, polynucleotide molecules encoding herbicide-resistant 5-enolpyruvir schimate-3-phosphate synthase (EPSPS) for conferring glyphosate resistance.

[0131] In some embodiments, glyphosate resistance can also be obtained by the expression of a polynucleotide molecule encoding glyphosate oxidoreductase (GOX) or glyphosate-N-acetyltransferase (GAT).

[0132] In some embodiments, polynucleotides encoding heterophosphinothricin acetyltransferases can be used for herbicide resistance. In some embodiments, plants containing heterophosphinothricin acetyltransferases can show improved resistance to glufosinate herbicides, for example, by inhibiting the enzyme glutamine synthetase.

[0133] In some embodiments, polynucleotides encoding proteins with modified protoporphyrinogen oxidase (PPO or PROTOX) activity can be used for herbicide resistance. In some embodiments, plants containing such polynucleotides can show improved resistance to any of the various herbicides that can target the PPO enzyme (also referred to as "PPO inhibitors" or "PROTOX inhibitors").

[0134] In some embodiments, dicamba monooxygenase can be used to confer dicamba resistance.

[0135] In some embodiments, polynucleotide molecules encoding AAD12 or AAD1 can be used, for example, to confer resistance to auxin herbicides.

[0136] In some embodiments, P450 coding polynucleotides can be used to confer herbicide resistance. In some embodiments, P450 coding sequences can confer resistance to HPPD inhibitors, for example, through herbicide metabolism. Such sequences include, but are not limited to, the NSF1 gene.

[0137] Resistance to plant pests In some embodiments, “plant pest” may mean any stage of life of any entity that can directly or indirectly damage, cause damage to, or cause disease to any plant or plant product. In some embodiments, plant pests may include protozoa, non-human animals, parasitic plants, bacteria, fungi, viruses, viroids, infectious agents, pathogens, or any similar or associated objects.

[0138] In some embodiments, plant-harmful invertebrates may include harmful nematodes, harmful mollusks, harmful insects, or any combination thereof. In some embodiments, harmful mollusks may include slugs, snails, or a combination thereof. In some embodiments, plant pathogens may include fungi, nematodes, or a combination thereof.

[0139] In some embodiments, the plant pathogen may be a eukaryotic plant pathogen. In some embodiments, the plant pathogen may be a fungal pathogen, such as a plant pathogenic fungus.

[0140] In some embodiments, the target gene of interest (for example, for gene silencing) can be any coding or non-coding sequence from any species (including, but not limited to, eukaryotes such as fungi, plants including monocots and dicots such as fungi, crop plants, ornamental plants, and uncultivated or wild plants, invertebrates such as arthropods, annelids, nematodes, and mollusks, and vertebrates such as amphibians, fish, birds, and mammals). In some embodiments, non-limiting examples of non-coding sequences (which can be expressed by gene expression elements such as regulatory sequences) can be 5' untranslated regions, promoters, enhancers, or other non-coding transcription regions, 3' untranslated regions, terminators, introns, microRNAs, microRNA precursor DNA sequences, small interfering RNAs, RNA components of ribosomes or ribozymes, small nucleolar RNAs, and other non-coding RNAs, or any combination thereof. In some embodiments, the gene of interest may include a translatable (coding) sequence, such as a gene encoding a transcription factor, and a gene encoding an enzyme involved in the biosynthesis or catabolism of the molecule of interest (such as amino acids, fatty acids and other lipids, sugars and other carbohydrates, biopolymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin).

[0141] In some embodiments, the target gene (e.g., for gene silencing) may be an essential gene of a plant pest or plant pathogen. In some embodiments, essential genes may include genes that are necessary for the development of the pest or pathogen into a reproductive adult. In some embodiments, essential genes may include genes that, when silenced or suppressed, result in the death of the organism (e.g., as an adult or at any developmental stage including gametes) or render normal reproduction of the organism impossible (e.g., sterility in male or female parents, or lethality to zygotes, embryos, or larvae).

[0142] In some embodiments, plants can be transformed (e.g., in the nucleus, cytoplasmic organelles, or both) by expression cassettes encoding, for example, dsRNA, siRNA, or miRNA. The dsRNA, siRNA, or miRNA can repress (e.g., the expression of) at least one (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) target genes present in a plant pest. In some embodiments, the dsRNA, siRNA, or miRNA can repress, for example, one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more target genes of a plant pest. In some embodiments, repression of target genes present in a plant pest can result in complete or near-complete protection from the plant pest. In some embodiments, "complete protection" may mean that (e.g., substantial) damage cannot be inflicted on the plant by the plant pest.

[0143] In some embodiments, resistance to pests in plants can be achieved, for example, by transgenic control. In some embodiments, for example, intraplant transgenic control of pests can be achieved, for example, by the plant expression of the crystalline (Cry) delta-endotoxin gene and / or plant insecticidal protein (VIP) (e.g., derived from Bacillus thuringiensis). In some embodiments, non-limiting examples of Cry toxins include, for example, 60 major groups of "Cry" toxins (e.g., Cry1-Cry59) and VIP toxins. In some embodiments, cry toxins may include subgroups of Cry toxins, for example, Cry1a.

[0144] In some embodiments, an expression cassette for use in transformation (e.g., into organelles) may be constructed using, for example, a Cry sequence. In some embodiments, the Cry sequence may include, for example, a wild-type (e.g., native) nucleic acid sequence encoding at least one protein selected from the group consisting of Cry1Ac, Cyt1Aa, Cry1Ab, Cry2Aa, Cry1I, Cry1C, Cry1D, Cry1E, Cry1Be, Cry1Fa, and Vip3A. In some embodiments, the Cry sequence may include, for example, a modified (e.g., cleaved or fused) nucleic acid sequence encoding at least one protein selected from the group consisting of Cry1Ac, Cyt1Aa, Cry1Ab, Cry2Aa, Cry1I, Cry1C, Cry1D, Cry1E, Cry1Be, Cry1Fa, and Vip3A. In some embodiments, the modified sequence may include a cleaved nucleic acid sequence. In some embodiments, the modified sequence may encode a modified protein fragment. In some embodiments, the cleaved protein fragments may retain insecticidal activity. In some embodiments, the nucleic acid sequence may encode a full-length or modified (e.g., cleaved) protein. In some embodiments, the modified protein may be codon-optimized for the organelle of interest.

[0145] Genome modification In some embodiments herein, compositions and methods are disclosed that may be used for genomic modification of target sequences in the genome (e.g., nuclear, plastid, or mitochondrial genome) of an organism or cell (e.g., plant or plant cell), for selection of a modified organism or cell, for gene editing, and for insertion of donor polynucleotides into the genome (e.g., nuclear, plastid, or mitochondrial genome) of an organism or cell. In some embodiments, the methods disclosed herein may utilize polynucleotide-derived polypeptide systems, such as guide polynucleotide / Cas protein systems. In some embodiments, the Cas protein may be induced by the guide polynucleotide to recognize a target polynucleic acid. In some embodiments, the Cas protein may introduce single-strand or double-strand breaks into the cell genome at a specific target site. In some embodiments, the guide polynucleotide / Cas polypeptide system may provide an effective system for modifying target sites in the genome of a plant, plant cell, or seed.

[0146] In some embodiments, various methods can be used to further modify the target site to introduce the desired donor polynucleotide. In some embodiments, the nucleotide sequence to be edited (e.g., the desired nucleotide sequence) may be located inside or outside the target site that can be recognized by the polynucleotide-derived polypeptide.

[0147] This specification also discloses methods and compositions utilizing polynucleotide-derived polypeptide systems for modifying multiple target sites within the organelle genome. Modification of multiple target sites within the organelle genome can facilitate the generation of homoplasmic transformation events.

[0148] Polynucleotide-derived polypeptide systems In some embodiments, the polynucleotide-derived polypeptide may be a polypeptide capable of binding to a target nucleic acid. In some embodiments, the polynucleotide-derived polypeptide may be a nuclease (e.g., a CRISPR nuclease). In some embodiments, the polynucleotide-derived polypeptide may be an endonuclease, a modified form thereof, or a biologically active fragment thereof. In some embodiments, the polynucleotide-derived polypeptide may be a Cas protein, a modified form thereof, or a biologically active fragment thereof. In some embodiments, the polynucleotide-derived polypeptide may be a MAD protein, a modified form thereof, or a biologically active fragment thereof. In some embodiments, the polynucleotide-derived polypeptide may be an Argonaut protein, a modified form thereof, or a biologically active fragment thereof. In some embodiments, the polynucleotide-derived polypeptide may form a complex with a guide polynucleotide. In some embodiments, the polynucleotide-derived polypeptide may be guided to the target nucleic acid by the guide polynucleotide. In some embodiments, the polynucleotide-derived polypeptide may form a complex with a guide polynucleotide to recognize the target nucleic acid. In some embodiments, the polynucleotide-derived polypeptide may introduce single-strand or double-strand disruption at a specific target site (e.g., the cellular genome).

[0149] In some embodiments, the polynucleotide-derived polypeptide may be a Cas protein in the CRISPR / Cas system. In some embodiments, the Cas protein may be a class 1 or class 2 Cas protein. In some embodiments, the Cas protein may be a type I, type II, type III, type IV, type V, or type VI Cas protein.

[0150] In some embodiments, non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Casl, Caslb, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas10, Cas10d, CaslO, CaslOd, CasF, CasG, CasH, Cpf1, Csyl, Csy2, Csy3, and Csel. Examples include (CasA), Cse2(CasB), Cse3(CasE), Cse4(CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, as well as their homologs or modified forms.

[0151] In some embodiments, the Cas protein may be derived from any suitable organism. In some embodiments, suitable organisms include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, and Alicyclobacillus acidocardarius. acidocaldarius), Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxydance ferrooxidans), Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.This may include *Microcoleus chthonoplastes*, species of the genus *Oscillatoria*, *Petrotoga mobilis*, *Thermosipho africanus*, *Acaryochloris marina*, *Leptotrichia shahii*, and *Francisella novicida*. In some embodiments, the organisms may include *Streptococcus pyogenes*.

[0152] In some embodiments, the Cas protein may comprise the Cas9 protein. In some embodiments, the Cas9 protein may comprise the Cas9 sequences listed in SEQ ID NOs. 462, 474, 489, 494, 499, 505, and 518 of International Publication WO2007 / 025097, which is incorporated herein by reference. In some embodiments, the Cas9 protein can unwind a double-stranded DNA adjacent to a genomic target site. In some embodiments, the Cas9 protein can cleave both DNA strands upon recognition of the target sequence by a guide polynucleic acid. In some embodiments, the Cas9 endonuclease can cleave only if the precise protospacer adjacent motif (PAM) is approximately oriented to the 3' end of the target sequence. In some embodiments, mutagenesis of the Streptococcus pyogenes Cas9 catalytic domain can produce a "nicking" enzyme (Cas9n) that can induce single-stranded nicks rather than double-stranded disruption.

[0153] In some embodiments, the polynucleotide-derived polypeptide may be a MAD polypeptide, e.g., MAD2 or MAD7 polypeptide, having amino acid sequences corresponding to Sequence ID No. 2 and Sequence ID No. 7 of U.S. Patent No. 9,982,279 (materially incorporated herein by reference). In some embodiments, MAD7 may be a class 2 VA-type CRISPR-Cas system isolated from Eubacterium rectale and reengineered by INSCRIPTA® (Boulder, CO). In some embodiments, similar to Cas9, MAD7 may be an RNA-induced nuclease with diverse protein structures, mechanisms of action, and demonstrated gene-editing activity in E. coli and yeast cells. In some embodiments, similar to Acidaminococcus sp. Cas12a, MAD7 does not require tracrRNA and prefers T-rich PAMs (TTTV and CTTV). In some embodiments, mutagenesis of MAD2 or MAD7 can produce a “nicking” enzyme that can induce single-strand nicks rather than double-strand disruption.

[0154] In some embodiments, the polynucleotide-derived polypeptide may be an Argonaut protein, such as Natronobacterium gregoryi Argonaute, "NgAgo". In some embodiments, the Argonaut protein may be a DNA-induced endonuclease. In some embodiments, the Argonaut protein may bind to guide DNA, such as a 24-nucleotide 5'-phosphorylated single-strand guide DNA (gDNA). In some embodiments, upon loading of gDNA, the Argonaut protein may cause site-directed target nucleic acid (e.g., DNA) disruption (e.g., double-strand disruption). In some embodiments, the Argonaut protein / gDNA system may not require a protospacer adjacent motif (PAM) for recognition of the target nucleic acid.

[0155] In some embodiments, the polynucleotide-derived polypeptide used herein may be the wild-type or modified form of the polynucleotide-derived polypeptide. In some embodiments, the polynucleotide-derived polypeptide may be an active variant, an inactive variant, or a fragment of the wild-type or modified polynucleotide-derived polypeptide. In some embodiments, the polynucleotide-derived polypeptide may include amino acid changes such as deletions, substitutions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof, compared to the wild-type form of the polynucleotide-derived polypeptide. In some embodiments, the polynucleotide-derived polypeptide may be a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity with an exemplary wild-type polynucleotide-derived polypeptide (e.g., Cas9 from Streptococcus pyogenes). In some embodiments, the polynucleotide-derived polypeptide may be a polypeptide having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or similarity to a wild-type exemplary polynucleotide-derived polypeptide. In some embodiments, the mutant or fragment may contain at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity to a wild-type or modified polynucleotide-derived polypeptide or its portion. In some embodiments, the mutant or fragment may be targeted to a nucleic acid locus that forms a complex with a guide nucleic acid, even though it lacks nucleic acid cleavage activity.

[0156] In some embodiments, the polynucleotide-derived endonucleases may be fusion proteins. In some embodiments, the polynucleotide-derived endonucleases may be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, non-limiting examples of suitable fusion partners include polypeptides that result in methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity, or any combination thereof. In some embodiments, the polynucleotide-derived endonucleases may also be fused to heterologous polypeptides that result in increased or decreased stability. In some embodiments, the fusion domain or heterologous polypeptide may be located at the N-terminus, C-terminus, or internally within the polynucleotide-derived endonucleases.

[0157] In some embodiments, nucleic acids encoding polynucleotide-derived endonucleases (e.g., Cas endonuclease, Cas9 endonuclease, MAD polypeptide, MAD7 polypeptide) can be codon-optimized for efficient translation into proteins in specific cells, organelles (e.g., nucleus, plastid, or mitochondria), or organisms (e.g., wheat or rice).

[0158] In some embodiments, nucleic acids encoding polynucleotide-derived endonucleases can be stably incorporated into the cell genome (nucleus, mitochondria, plastids). In some embodiments, nucleic acids encoding polynucleotide-derived polypeptides can be manipulably ligated to active regulatory sequences within the cell. In some embodiments, nucleic acids encoding polynucleotide-derived polypeptides may be present in expression constructs. In some embodiments, expression constructs may include any regulatory sequences that can lead to the expression of a desired nucleic acid sequence (promoter, terminator, RNA editing site). In some embodiments, expression constructs may include any nucleic acid sequence encoding a peptide capable of targeting a protein to a desired organelle (e.g., nucleus, mitochondria, or plastids).

[0159] In some embodiments, the polynucleotide-derived polypeptide coding sequence can be modified to use codons preferred by codon-optimized sequences of a target organism, e.g., plants, maize, or soybeans (nucleus, mitochondria, or plastids). In some embodiments, the sequence encoding the polynucleotide-derived polypeptide can be operably ligated to one or more sequences encoding nuclear localization signals, for example, an SV40 nuclear targeting signal upstream of the polynucleotide-derived polypeptide coding region and a bifurcation VirD2 nuclear localization signal downstream of the polynucleotide-derived polypeptide coding region. In some embodiments, the sequence encoding the polynucleotide-derived polypeptide can be operably ligated to one or more sequences encoding chloroplast or mitochondrial localization signals, i.e., chloroplast transit sequences or mitochondrial targeting sequences.

[0160] In some embodiments, polynucleotide-derived polypeptides (e.g., Cas polypeptide, Cas9 polypeptide, MAD polypeptide, MAD7 polypeptide) can be provided in any form. In some embodiments, polynucleotide-derived polypeptides can be provided in the form of proteins, such as polynucleotide-derived polypeptides alone or in complex with guide nucleic acids. In some embodiments, polynucleotide-derived polypeptides can be provided in the form of nucleic acids encoding the polynucleotide-derived polypeptide, such as RNA (e.g., messenger RNA (mRNA)) or DNA.

[0161] In some embodiments, a polynucleotide-derived polypeptide may be a polypeptide moiety (e.g., a chimeric polypeptide) that can form a programmable nucleoprotein complex with a specificity-constituting nucleic acid (SCNA). In some embodiments, the programmable nucleoprotein complex can be constructed in vivo, in target cells, or in organelles. In some embodiments, the programmable nucleoprotein complex can interact with a given target nucleic acid sequence. In some embodiments, the programmable nucleoprotein complex may include a polynucleotide molecule encoding a chimeric polypeptide. In some embodiments, the chimeric polypeptide may include a functional domain that can modify a target nucleic acid site. In some embodiments, the functional domain may lack a specific nucleic acid binding site. In some embodiments, the chimeric polypeptide may include a ligation domain that can interact with an SCNA. In some embodiments, the ligation domain may lack a specific target nucleic acid binding site. In some embodiments, the SCNA may include a nucleotide sequence complementary to a region of the target nucleic acid adjacent to the target site. In some embodiments, the SCNA may include a recognition region that can specifically bind to the ligation domain of the chimeric polypeptide. In some embodiments, a functional nuclear protein complex can be formed by constructing a chimeric polypeptide and SCNA within the target cell. In some embodiments, the nuclear protein complex can specifically modify the target nucleic acid at the target site.

[0162] In some embodiments, the polynucleotide-induced endonuclease gene may be a full-length polynucleotide-induced endonuclease (e.g., Cas endonuclease, Cas9 endonuclease, MAD polypeptide, MAD7 polypeptide), or any functional fragment or functional variant thereof.

[0163] In some embodiments of this specification, compositions and methods comprising the use of endonucleases are disclosed. In some embodiments, the endonucleases may be enzymes that cleave phosphodiester bonds within polynucleotide chains. In some embodiments, the endonucleases may include restriction endonucleases that cleave DNA at specific sites without damaging bases. In some embodiments, restriction endonucleases may include type I, type II, type III, and type IV endonucleases, and further may include their subtypes. In some embodiments, in the type I and type III systems, both methylase activity and restriction activity may be included in a single complex. In some embodiments, the endonucleases may also include meganucleases, also known as homing endonucleases (heases). In some embodiments, meganucleases can bind and cleave at specific recognition sites, which may be about 18 bp or larger. In some embodiments, meganucleases can be classified into four families based on conserved sequence motifs. In some embodiments, the meganuclease family may include the LAGLIDADG (SEQ ID NO: 1), GIY-YIG, HNH, and His-Cys box families. In some embodiments, motifs may be involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. In some embodiments, heases may have long recognition sites and may tolerate sequence polymorphisms in their DNA substrates. In some embodiments, the nomenclature rules for meganucleases may be similar to those for other restriction endonucleases.

[0164] In some embodiments, meganucleases may also be characterized by the enzyme prefix F-, I-, or PI-, encoded by an independent ORF, intron, and intein, respectively. In some embodiments, one step in the recombination process may involve polynucleotide cleavage at or near the recognition site. In some embodiments, cleavage activity can be used to cause double-strand disruption. In some embodiments, the recombinase may be derived from the integrase or resolvese family.

[0165] In some embodiments, the compositions and methods of the present disclosure may utilize transcription activator-like effector nucleases (TALENs, TAL effector nucleases). In some embodiments, TALENs may be a class of sequence-specific nucleases. In some embodiments, TALENs can be used to perform cleavage (e.g., double-strand disruption) at specific target sequences (e.g., those in the genome of plants or other organisms). In some embodiments, TALENs can be prepared by fusing a native or engineered transcription activator-like (TAL) effector, or a functional portion thereof, to the catalytic domain of an endonuclease, such as FokI. In some embodiments, a unique molecular TAL effector DNA-binding domain may enable the design of proteins that may have any given DNA recognition specificity.

[0166] In some embodiments herein, compositions and methods comprising the use of zinc finger nucleases (ZFNs) are disclosed. In some embodiments, the ZFN may be an engineered cleavage (e.g., double-strand disruption) inducer comprising a zinc finger DNA-binding domain and a double-strand disruption inducer domain. In some embodiments, recognition site specificity may be conferred by a zinc finger domain, which may include, for example, two, three, or four zinc fingers having a C2H2 structure. In some embodiments, the zinc finger domain may be suitable for designing polypeptides that specifically bind to a selected polynucleotide recognition sequence. In some embodiments, the ZFN may consist of an engineered DNA-binding zinc finger domain linked to a nonspecific endonuclease domain, for example, a nuclease domain derived from an IIS-type endonuclease such as FokI. In some embodiments, further functionality may be fused to the zinc finger-binding domain, which may include a transcription activator domain, a transcription repressor domain, and a methylase. In some examples, cleavage activity may require dimerization of the nuclease domain. In some embodiments, each zinc finger can recognize, for example, three consecutive base pairs in target DNA. In some embodiments, the 3-finger domain can recognize a sequence of nine consecutive nucleotides, and for the nuclease dimerization requirements, two sets of zinc finger triplets can be used to ligate an 18-nucleotide recognition sequence.

[0167] Guide polynucleotides In some embodiments, bacteria and archaea may possess a well-developed adaptive immune defense known as a clustered, regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system to guide the degradation of foreign nucleic acids. In some embodiments, bacterial type II CRISPR / Cas systems can utilize crRNA and tracrRNA to induce Cas polypeptides to nucleic acid targets. In some embodiments, crRNA (CRISPR RNA) may contain a region complementary to one strand of the double-stranded DNA target. In some embodiments, crRNA can base-pair with tracrRNA (trans-activated CRISPR RNA) to form an RNA double-strand that allows the Cas polypeptide to recognize the DNA target and selectively cleave it.

[0168] In some embodiments, as used herein, the term “guide polynucleotide” may refer to a polynucleotide sequence that can form a complex with a polynucleotide-derived polypeptide (e.g., Cas protein, MAD protein). In some embodiments, the guide polynucleotide can cause the polynucleotide-derived polypeptide to recognize a DNA target site and optionally cleave (or nicking) it. In some embodiments, the terms “guide polynucleotide” and “guide polynucleic acid” may be used interchangeably herein. In some embodiments, the guide polynucleotide may consist of a single molecule (monomolecular) or two molecules (bimolecular). In some embodiments, the guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (a combination of RNA-DNA sequences). In some embodiments, a guide polynucleotide that can contain ribonucleic acid on its own may also be referred to as “guide RNA” (gRNA). In some embodiments, the guide polynucleic acid may be a guide RNA.

[0169] In some embodiments, the term “single guide RNA” (sgRNA) may refer to a synthetic fusion of two RNA molecules, for example, a crRNA (CRISPR RNA) and a tracrRNA containing a variable targeting domain. In some embodiments, the guide RNA may comprise a variable targeting domain (i.e., a VT domain) of 12 to 30 nucleotides, and an RNA fragment that can interact with the Cas protein.

[0170] In some embodiments, the guide polynucleotide may be a dimorphic molecule (i.e., two molecules, also referred to as a "dual molecule," "dual," or "double-stranded" guide polynucleotide) comprising, for example, a first molecule having a nucleotide sequence domain complementary to the nucleotide sequence in the target polynucleic acid (e.g., target DNA) (referred to as a variable targeting domain, or VT domain), and a second molecule having a nucleotide sequence domain that interacts with the Cas polypeptide (referred to as a Cas endonuclease recognition domain, or CER domain).

[0171] In some embodiments, the complementarity between the guide polynucleic acid (e.g., VT domain, spacer region) and the target polynucleic acid (e.g., protospacer) may be complete, substantial, or sufficient. In some embodiments, complete complementarity between two nucleic acids may mean that the two nucleic acids can form a double helix in which all bases in the double helix can be bound to complementary bases by Watson-Crick pairing. In some embodiments, substantial or sufficient complementarity may mean that the sequence in one strand may not be completely and / or perfectly complementary to the sequence in the opposing strand, but sufficient binding occurs between the bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature).

[0172] In some embodiments, the terms “variable targeting domain” or “VT domain” may be used interchangeably herein and may refer to a nucleotide sequence that may be present in a guide polynucleotide. In some embodiments, the VT domain may be complementary to one strand of the double-stranded DNA target site. In some embodiments, the complementarity percentage between the first nucleotide sequence domain (VT domain) and the target sequence may be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the variable targeting domain may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the variable targeting domain may contain at least 17 nucleotides complementary to at least 17 nucleotides of the target polynucleic acid. In some embodiments, the variable targeting domain may contain a contiguous stretch of nucleotides complementary to the target polynucleic acid. In some embodiments, the nucleotides of the guide polynucleic acid complementary to the target polynucleic acid may be discontinuous. In some embodiments, the variable targeting domain may contain a contiguous stretch of 12 to 30 nucleotides. In some embodiments, the variable targeting domain may consist of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0173] In some embodiments, the nucleotide sequence linking the cr nucleotide and tracr nucleotide of the single guide polynucleotide may include an RNA sequence, a DNA sequence, or a combination of RNA-DNA sequences. In some embodiments, the nucleotide sequence linking the cr nucleotide and tracr nucleotide of the single guide polynucleotide may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, The lengths may be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. In some embodiments, the nucleotide sequence linking the cr nucleotide and tracr nucleotide of the single guide polynucleotide may include, but is not limited to, a GAAA tetranucleotide loop sequence.

[0174] In some embodiments, guide polynucleotides can be introduced into plant cells by transformation of a recombinant DNA construct containing a polynucleotide encoding a guide polynucleotide that is operably linked to a functional promoter in plants, such as a plant U6 polymerase III promoter, a CaMV 35S polymerase II promoter, a mitochondrial promoter, or a plastid promoter.

[0175] In some embodiments, multiple guide polynucleic acids can be multiplexed to target multiple target nucleic acids. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 target nucleic acids can be targeted simultaneously or repeatedly.

[0176] Target sites for genome modification In some embodiments, the terms “target site,” “target sequence,” “target polynucleotide,” “target polynucleic acid,” “target locus,” “genomic target site,” “genomic target sequence,” and “genomic target locus” may be used interchangeably herein. In some embodiments, the target polynucleic acid may refer to a polynucleotide sequence within a genome (e.g., a plastid or mitochondrial genome). In some embodiments, the genome may be part of a plant cell. In some embodiments, the target polynucleic acid may refer to a site recognized by a guide polynucleic acid (e.g., one within a genome). In some embodiments, the target polynucleic acid may refer to a site (e.g., one within a genome) that can induce single-stranded or double-stranded disruption (e.g., by a Cas polypeptide). In some embodiments, the target site may be an endogenous site within a genome. In some embodiments, the target site may be heterogeneous to the organism and therefore not naturally present in the genome. In some embodiments, the target site may be found at a heterogeneous genomic location compared to its naturally occurring location. In some embodiments, the terms “endogenous target sequence” and “natural target sequence,” as used herein, may be interchangeable herein and may refer to a target sequence that may be endogenous or natural to the genome of an organism. In some embodiments, the endogenous target sequence may occur at an endogenous or native location within the organism's genome.

[0177] In some embodiments, the target polynucleic acid may be DNA, RNA, or both. In some embodiments, the target polynucleic acid may be DNA (e.g., target DNA). In some embodiments, the target polynucleic acid may be genomic DNA. In some embodiments, the target polynucleic acid may be nuclear DNA, mitochondrial DNA, plastid DNA, or any combination thereof.

[0178] In some embodiments, the terms “artificial target site” and “artificial target sequence” may be used interchangeably herein and may refer to a target sequence introduced into the genome of a plant. In some embodiments, such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the organism, but may be located at a different location in the genome of the organism (i.e., a non-endogenous or non-native location).

[0179] In some embodiments, “modified target site,” “modified target sequence,” “modified target site,” and “modified target sequence” can be used interchangeably herein and may refer to a target sequence disclosed herein that may include at least one modification compared to an unmodified target sequence. In some embodiments, such “modification” may include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i) to (iii).

[0180] In some embodiments, the length of the target site can vary and may include, for example, a target site with a nucleotide length of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more. In some embodiments, the target site may be recursive. In some embodiments, the recursive sequence may include a sequence that reads the same thing in the opposite direction on the complementary strand in one strand. In some embodiments, the nicking / cleavage site may be within the target sequence. In some embodiments, the nicking / cleavage site may be outside the target sequence. In some embodiments, cleavage may occur at nucleotide positions directly opposite each other to result in a blunt-end cleavage, or cleavage may be alternately performed to produce a single-stranded overhang, also called a "sticky end," which may be either a 5' overhang or a 3' overhang.

[0181] In some embodiments, the target nucleic acid sequence may be 5' or 3' of the PAM. In some embodiments, the target nucleic acid sequence may be, for example, 16, 17, 18, 19, 20, 21, 22, or 23 bases located immediately around 5' of the first nucleotide of the PAM. In some embodiments, the target nucleic acid sequence may be 16, 17, 18, 19, 20, 21, 22, or 23 bases located immediately around 3' of the last nucleotide of the PAM. In some embodiments, the target nucleic acid sequence may be 20 bases located immediately around 5' of the first nucleotide of the PAM. In some embodiments, the target nucleic acid sequence may be 20 bases located immediately around 3' of the last nucleotide of the PAM.

[0182] In some embodiments, site-specific cleavage of a target nucleic acid by a polynucleotide-derived polypeptide (e.g., Cas protein) may occur at a location determined by base pair complementarity between the guide nucleic acid and the target nucleic acid. In some embodiments, site-specific cleavage of a target nucleic acid by a polynucleotide-derived polypeptide (e.g., Cas protein) may occur at a location determined by a protospacer adjacent motif (PAM). In some embodiments, the cleavage site of Cas (e.g., Cas9) may be about 1 to about 25, or about 2 to about 5, or about 19 to about 23 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some embodiments, the cleavage site of Cas (e.g., Cas9) may be 3 base pairs upstream of the PAM sequence. In some embodiments, the cleavage site of Cas (e.g., Cpf1) may be 19 bases on the (+) strand and 23 bases on the (-) strand, producing a 5' overhang of length 5 nt. In some cases, the cleavage may produce a blunt end. Depending on the circumstances, the cut may produce alternating or adhesive ends with a 5' overhang. Depending on the circumstances, the cut may produce alternating or adhesive ends with a 3' overhang.

[0183] In some embodiments, various organisms may contain various PAM sequences. In some embodiments, various Cas proteins may recognize various PAM sequences. In some embodiments, in Streptococcus pyogenes, the PAM may be a sequence in the target nucleic acid that may contain the sequence 5'-NRR-3', in which case R may be either A or G, N may be any nucleotide, and N may be immediately 3' of the target nucleic acid sequence targeted by the spacer sequence. In some embodiments, the PAM sequence of Streptococcus pyogenes Cas9 (SpyCas9) may be 5'-NGG-3', in which case N may be any DNA nucleotide and may be immediately 3' of the CRISPR recognition sequence on the non-complementary strand of the target DNA. In some embodiments, the PAM of Cpf1 may be 5'-TTN-3', in which case N may be any DNA nucleotide and may be immediately 5' of the CRISPR recognition sequence.

[0184] In some embodiments, consensus PAM sequences for various MAD polypeptides have been determined (U.S. Patent No. 9982279). In some embodiments, the consensus PAM for MAD1-MAD8 and MAD10-MAD12 was determined to be TTTN. In some embodiments, the consensus PAM for MAD9 was determined to be NNG. In some embodiments, the consensus PAM for MAD13-MAD15 was determined to be TTN. In some embodiments, the consensus PAM for MAD16-MAD18 was determined to be TA. In some embodiments, the consensus PAM for MAD19-MAD20 was determined to be TTCN.

[0185] In some embodiments, active variants of genomic target sites may also be used. In some embodiments, the active variant may contain at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to a given target site. In some embodiments, the active variant may retain biological activity. In some embodiments, the active variant may be recognized by a polynucleotide-derived polypeptide (e.g., Cas protein). In some embodiments, the active variant may be cleaved by a polynucleotide-derived polypeptide (e.g., Cas protein). In some embodiments, an assay may be used to measure double-strand disruption of the target site by an endonuclease. In some embodiments, an assay may be used to measure the overall activity and / or specificity of an endonuclease on a DNA substrate containing the recognition site (e.g., target site, active variant).

[0186] Method for incorporating donor polynucleotides In some embodiments, this disclosure provides a method for obtaining an organelle (e.g., mitochondria or plastids) containing a donor polynucleotide. In some embodiments, the method can utilize homologous recombination to bring about the incorporation of a polynucleotide at a target site. In some embodiments, homologous recombination can be enhanced by introducing double-strand breaks (DSBs) at a selected endonuclease target site. In some embodiments, this specification describes the use of a polynucleotide-derived polypeptide system that can provide flexible genome cleavage specificity and bring about high-frequency double-strand breaks at organelle DNA target sites. In some embodiments, specific cleavage can enable efficient gene editing of a desired nucleotide sequence. In some embodiments, the desired nucleotide sequence to be edited may be located inside or outside a target site recognized and / or cleaved by a polynucleotide-derived polypeptide (e.g., Cas polypeptide, MAD polypeptide).

[0187] In some embodiments, the polynucleotide of interest may be provided to an organelle in a donor polynucleotide. In some embodiments, the donor polynucleotide may be a nucleic acid sequence (e.g., DNA, RNA, or both) that can be incorporated into the genome of a target nucleic acid, such as a mitochondrial or plastid. In some embodiments, the donor polynucleotide may include a polynucleotide encoding a variant of a naturally occurring polypeptide having enzymatic activity (e.g., herbicide-resistant ALS, herbicide-resistant EPSPS, or herbicide-resistant GS). In some embodiments, the donor polynucleotide may include a polynucleotide encoding a variant of a naturally occurring polypeptide having enzymatic activity, and an additional polynucleotide encoding the gene of interest. In some embodiments, the gene of interest may be a cytoplasmic male sterility (CMS) coding region. In some embodiments, the donor polynucleotide may be inserted into the genome, for example, at a cleavage site of a polynucleotide-derived polypeptide. In some embodiments, the donor polynucleotide may be inserted into the genome by homologous recombination. In some embodiments, the method further includes the step of removing the polynucleotide encoding the variant of a naturally occurring polypeptide having enzymatic activity after the integration of the gene of interest. In some embodiments, the donor polynucleotide may include DNA and may be referred to as donor DNA.

[0188] In some embodiments, donor polynucleotides of any suitable size can be incorporated into the genome. In some embodiments, the donor polynucleotides incorporated into the genome may be less than 1 kb, about 1 kb, about 1.5 kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, about 10 kb, about 10.5 kb, about 11 kb, about 11.5 kb, about 12 kb, about 12.5 kb. It may be kb, approximately 13kb, approximately 13.5kb, approximately 14kb, approximately 14.5kb, approximately 15kb, approximately 16kb, approximately 17kb, approximately 18kb, approximately 19kb, approximately 20kb, approximately 25kb, approximately 30kb, approximately 35kb, approximately 40kb, approximately 45kb, approximately 50kb, approximately 100kb, approximately 150kb, approximately 200kb, approximately 250kb, approximately 300kb, approximately 350kb, approximately 400kb, approximately 450kb, approximately 500kb, or less than approximately 500 kilobases (kb). In some embodiments, the donor polynucleotides incorporated into the genome have lengths of at least approximately 1kb, approximately 1kb, approximately 1.5kb, approximately 2kb, approximately 2.5kb, approximately 3kb, approximately 3.5kb, approximately 4kb, approximately 4.5kb, approximately 5kb, approximately 5.5kb, approximately 6kb, approximately 6.5kb, approximately 7kb, approximately 7.5kb, approximately 8kb, approximately 8.5kb, approximately 9kb, approximately 9.5kb, approximately 10kb, approximately 10.5kb, approximately 11kb, approximately 11.5kb, approximately 12kb, and approximately 12.5kb. It may be kb, approximately 13kb, approximately 13.5kb, approximately 14kb, approximately 14.5kb, approximately 15kb, approximately 16kb, approximately 17kb, approximately 18kb, approximately 19kb, approximately 20kb, approximately 25kb, approximately 30kb, approximately 35kb, approximately 40kb, approximately 45kb, approximately 50kb, approximately 100kb, approximately 150kb, approximately 200kb, approximately 250kb, approximately 300kb, approximately 350kb, approximately 400kb, approximately 450kb, approximately 500kb, or less than approximately 500 kilobases (kb).In some embodiments, the donor polynucleotides incorporated into the genome are up to approximately 1kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 10.5kb, 11kb, 11.5kb, and 12kb in length. b. It could be approximately 12.5kb, 13kb, 13.5kb, 14kb, 14.5kb, 15kb, 16kb, 17kb, 18kb, 19kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, or up to approximately 500kb.

[0189] In some embodiments, the donor polynucleotide may include the polynucleotide of interest, a polynucleotide modification template, a heterologous expression cassette, or any combination thereof. In some embodiments, the term “polynucleotide modification template” may refer to a polynucleotide that may contain at least one nucleotide modification compared to the nucleotide sequence to be edited. In some embodiments, the nucleotide modification may be at least one nucleotide substitution, addition, deletion, or any combination thereof. In some embodiments, minor genomic modifications produced by the use of a polynucleotide modification template may include the production of a mutant allele (e.g., an antibiotic resistance rRNA gene) and the removal of a target site in a polynucleotide-derived polypeptide. In some embodiments, the donor polynucleotide (e.g., donor DNA) may include first and second homologous regions. In some embodiments, the donor polynucleotide includes heterologous sequences adjacent to the first and second homologous regions. In some embodiments, the first and second homologous regions of the donor polynucleotide (e.g., donor DNA) may share homology with first and second genomic regions that are present in or adjacent to the target site (e.g., one in an organelle genome), respectively.

[0190] In some embodiments, “homology” may mean similar DNA sequences. In some embodiments, homology may mean nucleic acid sequences having, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity. In some embodiments, a “homology region to a genomic region” may be a DNA region having a sequence similar to a given “genomic region” in the organelle genome. In some embodiments, the homology region may be of any length that is sufficient to promote homologous recombination at the cleaved target site. In some embodiments, the homology region may contain at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, or more bases in length, so that it can have sufficient homology to undergo homologous recombination with the corresponding genomic region. In some embodiments, “sufficient homology” may indicate that two polynucleotide sequences can have sufficient structural similarity to act as substrates for homologous recombination. In some embodiments, the donor polynucleotide (e.g., donor DNA) may include an expression cassette (e.g., one encoding the heterologous polynucleotide of interest). In some embodiments, the donor polynucleotide may include multiple expression cassettes. In some embodiments, the expression cassette may be a polycistronic expression cassette in which, for example, multiple protein-coding regions, functional RNAs, or a combination of both are expressed under the control of a single promoter.

[0191] In some embodiments, the method may further include the steps of introducing a polynucleotide containing a Rep coding region into the nucleus of a cell and introducing a VOR-donor-VOR polynucleotide into the mitochondria of a cell. In some embodiments, a modified Rep protein, including a Rep protein operably linked to a mitochondrial-targeting peptide, can be introduced into the nucleus of a cell. In some embodiments, the modified Rep protein may contain an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 111. In some embodiments, a polynucleotide encoding a Rep protein or a modified Rep protein (e.g., nMTS-Rep) can be operably linked to an inducible promoter. In some embodiments, a polynucleotide encoding a Rep protein or a modified Rep protein can be operably linked to a constitutively active promoter.

[0192] In some embodiments, the VOR-donor DNA-VOR polynucleotide may include a geminiviral VOR sequence such that 5' and 3' donor DNA regions having sequences homologous to the target site are adjacent to the VOR sequence, resulting in VOR-donor DNA-VOR coordination. In some embodiments, the VOR sequence may include a polynucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 71.

[0193] In some embodiments, the Rep protein can induce replication of donor DNA adjacent to the VOR sequence, which is the target site of the geminiviral Rep protein. In some embodiments, amplification of donor DNA mediated by the Rep-VOR interaction increases the gene expression of the donor DNA by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, or 500% compared to the gene expression of donor DNA lacking the VOR element, the Rep protein, or both, after transformation into an organelle.

[0194] In some embodiments, “donor RNA” may contain, for example, the same nucleic acid sequence as donor DNA, i.e., it may be a corresponding RNA molecule having uridilate ("U") instead of deoxythymidylate ("T"). In some embodiments, “donor polynucleotide” may be either donor DNA or donor RNA, or a combination of DNA and RNA. In some embodiments, donor polynucleotide may be single-stranded or double-stranded.

[0195] In some embodiments, an alternative method for modifying the organelle genome may be to replace part or all of the organelle DNA with “replacement DNA”. In some embodiments, endogenous organelle DNA can be reduced or eliminated by the use of site-specific endonucleases such as polynucleotide-derived polypeptides (e.g., Cas polypeptide, Cas9 polypeptide, MAD polypeptide, MAD7 polypeptide). In some embodiments, replacement DNA can be introduced simultaneously or afterward. In some embodiments, the term “replacement DNA” may refer to organelle DNA or a fragment of complete organelle DNA that, when transformed into an organelle, can transmit a new genotype and corresponding traits. In some embodiments, the terms “replacement DNA” and “replacement organelle DNA” may be used interchangeably herein. In some embodiments, in the case of organelle DNA fragments, they can be incorporated into the remaining endogenous organelle DNA by homologous recombination. In the case of complete organelle DNA replacement, the replacement DNA can be isolated from cultivars, strains, subspecies, and other species having a different DNA composition from the endogenous organelle DNA of the recipient cell. In some embodiments, the substituted DNA can also be partially and / or completely synthesized in vitro. In some embodiments, the substituted DNA may include both native and non-native sequences. In some embodiments, the substituted DNA may be linear DNA with repeat sequences at its ends when prepared in vitro. In some embodiments, the repeat sequences may be direct repeats or inverted repeats. In some embodiments, the ends may facilitate homologous recombination in vitro or in vivo to produce circular DNA for replication of organelle DNA in cells. In some embodiments, the DNA prepared in vitro may also include heterologous DNA elements, such as those that enable selected amplification in bacterial cells. In some embodiments, the substituted DNA may include DNA elements that function as DNA replication origins in the recipient organelle. In some embodiments, the substituted DNA may include multiple DNA fragments capable of undergoing recombination within the organelle to result in complete substituted DNA.

[0196] In some embodiments, sequences that function as origins of replication may be included with the compositions of the Disclosure (e.g., polynucleotides, constructs, cassettes). Such sequences may include organelle origins of replication. In some embodiments, the origin of replication sequence may be a plastid origin of replication sequence (e.g., a plastid rRNA intergenetic region). In some embodiments, the origin of replication sequence may be a mitochondrial origin of replication sequence.

[0197] In some embodiments, as used herein, “genomic region” may refer to a DNA segment in the genome of an organelle (e.g., a mitochondrion or a plastid). In some embodiments, the genomic region may be present on both sides of the target site. In some embodiments, the genomic region may comprise a portion of the target site. In some embodiments, the genomic region may contain at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, or more bases. In some embodiments, the genomic region may contain sufficient homology to undergo homologous recombination with a corresponding homology region present on the donor DNA.

[0198] In some embodiments, the donor polynucleotide, the polynucleotide of interest, and / or the trait can be stacked integrally at a complex trait locus. In some embodiments, a guide polynucleotide / polypeptide system can be used to induce double-strand disruption and stack the trait at a complex trait locus.

[0199] In some embodiments, two or more polynucleotides encoding RNA and / or proteins may be contained in a cassette as a polycistron unit. In some embodiments, the RNA-encoding polynucleotides may be expressed from separate cassettes.

[0200] In some embodiments, a guide polynucleotide / polypeptide system can be used to introduce one or more donor polynucleotides or one or more desired traits into one or more target sites by providing plant cells with one or more guide polynucleotides, one or more polynucleotide-derivative polypeptides (e.g., Cas polypeptide, MAD polypeptide), and optionally one or more donor polynucleotides (e.g., donor DNA). In some embodiments, organisms can be produced from cells that may contain alterations to the above one or more target sites of organelle DNA (e.g., mitochondrial DNA or plastid DNA), the alterations may be selected from the group consisting of (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, and (iv) any combination of (i) to (iii).

[0201] In some embodiments, the structural similarity between a given genomic region and the corresponding homology region of a donor polynucleotide (e.g., donor DNA) can be any degree of sequence identity that allows homologous recombination to occur. In some embodiments, the amount of homology or sequence identity shared by the “homologous region” of the donor polynucleotide (e.g., donor DNA) and the “genomic region” of the plant genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity that allows the sequence to undergo homologous recombination.

[0202] In some embodiments, a homology region of a donor polynucleotide (e.g., donor DNA) may have homology to any sequence adjacent to the target site. In some embodiments, the homology region may share significant sequence homology to a genomic sequence directly adjacent to the target site, but the homology region can be designed to have sufficient homology to a region that may be further 5' or 3' to the target site. In other embodiments, the homology region may further have homology to a fragment of the target site together with downstream genomic regions. In one embodiment, the first homology region may further include a first fragment of the target site, and the second homology region may include a second fragment of the target site, where the first and second fragments are distinct.

[0203] In some embodiments, as used herein, “homologous recombination” may refer to the exchange of DNA fragments between two DNA molecules at a homologous site. In some embodiments, the frequency of homologous recombination may be influenced by several factors. In some embodiments, the length of the homologous region may affect the frequency of homologous recombination events; for example, the longer the homologous region, the higher the frequency of homologous recombination may be. In some embodiments, the length of the homologous region required to observe homologous recombination may vary between species.

[0204] In some embodiments, intermolecular recombination may occur in mitochondria and plastids; for example, plants having transformed mitochondrial DNA or transformed plastid DNA may result from site-specific integration of foreign sequences through homologous recombination with adjacent sequences on the transformation vector.

[0205] In some embodiments, intramolecular recombination between repeating sequences may result in, for example, inversion if the repeats are recurrent, or deletion if they are direct.

[0206] In some embodiments, insertions can be targeted using endogenous mitochondrial sequences or plastid sequences to achieve efficient incorporation of foreign sequences by homologous recombination. In some embodiments, a positive correlation may exist between the recombination rate and the length and / or degree of sequence homology.

[0207] In some embodiments, the minimum adjacent sequence length in homologous recombination with the organelle genome may be affected by the introduction of single-stranded or double-stranded breaks (or both) in the organelle genome, such as polynucleotide-derived polypeptides.

[0208] In some embodiments, the efficiency of the disclosed methods for manipulating or modifying genomes may be at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

[0209] In some embodiments, the method may include the step of introducing a donor polynucleotide (e.g., donor DNA), a guide polynucleotide (or multiple guide polynucleotides), and a polynucleotide-derived polypeptide into an organelle (e.g., mitochondria or plastids) of a cell (e.g., a plant cell). In some embodiments, at least one single-strand or double-strand break can be introduced into a target site by the polynucleotide-derived polypeptide, and the first and second homology regions of the donor polynucleotide (e.g., donor DNA) may undergo homologous recombination with their corresponding homologous genomic regions, resulting in DNA exchange between the donor and the genome. In some embodiments, the method disclosed herein can incorporate all or part of the donor polynucleotide (e.g., donor DNA) into a single-strand or double-strand break at a target site in the organelle genome, thereby modifying the original target site and producing a modified genomic target site.

[0210] In some embodiments, the cells may be eukaryotic cells. In some embodiments, the cells may include human cells, animal cells, non-human animal cells, bacterial cells, fungal cells, insect cells, plant cells, protist cells, yeast cells, algal cells, or any combination thereof. In some embodiments, the cells may include wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, and soybean cells. In some embodiments, the cells may be part of an organism or tissue. In some embodiments, the organism may include plants, transgenic plants, or parts thereof, including cells, tissues, reproductive materials, seeds, pollen, offspring, or any combination thereof, produced by the methods described herein. In some embodiments, the cells may be isolated and purified human cells.

[0211] In some embodiments, the nucleotide to be edited may be located inside or outside the target site recognized and cleaved by the polynucleotide-derived polypeptide. In some embodiments, at least one nucleotide modification may not be a modification at the target site recognized and cleaved by the polynucleotide-derived polypeptide. In some embodiments, there may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900, or 1000 nucleotides between the at least one nucleotide to be edited and the organelle DNA target site. In some embodiments, the nucleotide to be edited may be located both inside and outside the target site (or multiple target sites) recognized and cleaved by the polynucleotide-derived polypeptide.

[0212] In some embodiments, the donor polynucleotide may include donor DNA. In some embodiments, the donor polynucleotide can be introduced by any suitable means. In some embodiments, a plant having a target site can be provided. In some embodiments, the donor polynucleotide (e.g., donor DNA) can be provided by any suitable transformation method, including, for example, Agrobacterium-mediated transformation or particulate gun bombardment. In some embodiments, the donor polynucleotide (e.g., donor DNA) may be transiently present in the cell or introduced via a viral replicon. In some embodiments, the donor polynucleotide (e.g., donor DNA) can be inserted into the organelle genome in the presence of a guide polynucleotide (e.g., guide RNA), a polynucleotide-derivable polypeptide (e.g., Cas polypeptide, MAD polypeptide), and a target site.

[0213] Target polynucleotides for integration at the target site In some embodiments, methods are further provided for identifying at least one plant cell comprising an organelle containing a genome with the incorporated polynucleotide of interest at a target site. In some embodiments, the organelle may include mitochondria, plastids, or a combination thereof. In some embodiments, the donor polynucleotide may contain the polynucleotide of interest. In some embodiments, various methods can be used to identify plant cells in which the genome has been inserted at or near the target site without using a screenable marker phenotype. In some embodiments, methods including but not limited to PCR, sequencing, nuclease digestion, Southern blotting, and any combination thereof can be considered as directly analyzing the target sequence to detect any changes in the target sequence.

[0214] In some embodiments, the method may also include the step of recovering a plant from plant cells containing the polynucleotide of interest incorporated into the organelle genome. In some embodiments, the plant may be sterile or reproductive.

[0215] In some embodiments, the polynucleotide or polypeptide of interest may include herbicide-resilience coding sequences, insecticidal coding sequences, nematode-resilience coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic stress-resilience coding sequences, biological stress-resilience coding sequences, sequences that modify plant traits, or any combination thereof. In some embodiments, plant traits may include yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and / or utilization, and oil content and / or composition, or any combination thereof. In some embodiments, the polynucleotide of interest may include genes that improve crop yield, polypeptides that improve crop desirability, proteins that confer resistance to abiotic stresses such as drought, nitrogen, temperature, salinity, toxic metals, or trace elements, or proteins that confer resistance to biological stresses such as toxins such as insecticides and herbicides, or attacks by fungi, viruses, bacteria, insects, and nematodes, and the development of diseases associated with these organisms. In some embodiments, the target gene may include, for example, information-related genes such as zinc fingers, communication-related genes such as kinases, and housekeeping-related genes such as heat shock proteins. In some embodiments, the target polynucleotide may include genes encoding traits important to agronomy, insect resistance, disease resistance, herbicide resistance, reproductive or sterility, grain characteristics, commercial products, or any combination thereof. In some embodiments, the target gene may include genes involved in oil, starch, carbohydrate, or nutrient metabolism, genes affecting photosynthesis, photorespiration, and ATP metabolism, or any combination thereof.

[0216] In some embodiments, commercial traits can also be obtained by the expression of proteins encoded on polynucleotides. In some embodiments, the commercial use of transformed plants may be the production of polymers and bioplastics. In some embodiments, the polynucleotide of interest may include genes encoding proteins such as β-ketothiolase, PHBase (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase, which can facilitate the expression of polyhydroxyalkanoates (PHAs). In some embodiments, commercial use may be the expression of genes that can increase starch for ethanol production.

[0217] In some embodiments, polynucleotides or polypeptides that may influence amino acid biosynthesis include, for example, anthranilate synthase (AS;EC 4.1.3.27) that can catalyze a first reaction that branches off from the aromatic amino acid pathway to tryptophan biosynthesis in plants, fungi, and bacteria. In some embodiments, in plants, the chemical process for tryptophan biosynthesis can be compartmentalized in chloroplasts. In some embodiments, the additional donor sequence of interest may include chorismate pyruvate lyase (CPL), which may refer to a gene encoding an enzyme that can catalyze the conversion of chorismate to pyruvate and pHBA. In some embodiments, the CPL gene may be derived from E. coli. In some embodiments, the CPL gene may have GenBank accession number M96268.

[0218] In some embodiments, the polynucleotide sequence of interest may encode a protein involved in providing disease or pest resistance. In some embodiments, “disease resistance” or “pest resistance” may enable a plant to at least partially avoid adverse symptoms or outcomes resulting from plant-pathogen interactions. In some embodiments, a pest resistance gene may encode resistance to pests with high yield resistance. In some embodiments, pests with high yield resistance may include rootworms, cutworms, European corn borers, or any combination thereof. In some embodiments, a disease or insect resistance gene may include lysozyme, cecropine, or any combination thereof. In some embodiments, a disease or insect resistance gene may result in antimicrobial defense, antifungal defense, nematode defense, insect defense, or any combination thereof. In some embodiments, an antifungal resistance gene or protein may include defensins, glucanases, chitinases, or any combination thereof. In some embodiments, nematode or insect defense genes or proteins may include Bacillus thuringiensis endotoxin, protease inhibitors, collagenases, lectins, glycosidases, or any combination thereof. In some embodiments, genes encoding disease resistance traits may include detoxification genes. In some embodiments, detoxification genes may include fumonisin genes, non-pathogenic (avr) genes, disease resistance (R) genes, or any combination thereof. In some embodiments, insect resistance genes may encode resistance to high-yield resistance pests such as rootworms, armyworms, European corn borers, or any combination thereof. In some embodiments, insect resistance genes may include Bacillus thuringiensis (Bt) toxic protein genes.

[0219] In some embodiments, the transgene, recombinant DNA molecule, DNA sequence of interest, or donor polynucleotide may comprise one or more DNA sequences for gene silencing of the target gene. In some embodiments, the target gene may comprise a plant pest gene or a plant pathogen gene. In some embodiments, the method for gene silencing may comprise the expression of the DNA sequence in a plant. In some embodiments, the method for gene silencing may comprise co-repression, antisense repression, double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA) interference, intron-containing hairpin RNA (ihpRNA) interference, transcription gene silencing, and microRNA (miRNA) interference.

[0220] In some embodiments, a reproductive plant may be a plant that can produce viable male and female gametes and may also be self-reproductive. In some embodiments, a self-reproductive plant may produce offspring plants without the contribution of any other plant of gametes and the genetic material contained therein. In some embodiments herein, methods are disclosed that include the use of plants that may not be self-reproductive. In some embodiments, a plant may not produce viable or otherwise reproductive male gametes, female gametes, or both. In some embodiments, as used herein, a “male-sterile plant” may be a plant that does not produce viable or otherwise reproductive male gametes. In some embodiments, as used herein, a “female-sterile plant” may be a plant that does not produce viable or otherwise reproductive female gametes. In some embodiments, male-sterile plants and female-sterile plants may be female-sterile and male-sterile, respectively. In some embodiments, male-sterile (or otherwise female-sterile) plants can produce viable offspring when crossed with female-sterile plants. In some embodiments, female-sterile (or otherwise male-sterile) plants can produce viable offspring when crossed with male-sterile plants. In some embodiments, the use of hybrid plants in certain crop species has been shown to dramatically increase crop yields. In some embodiments, hybrid crop systems may require a male-sterile line that can function as a female parent producing hybrid seeds through propagation by a pollen donor plant. In some embodiments, a method of transmitting male sterility without manual or mechanical intervention may involve the use of a cytoplasmic male sterility (CMS) gene. In some embodiments, the CMS gene may include a nucleic acid. In some embodiments, the CMS gene may include a heterologous nucleic acid. In some embodiments, the nucleic acid may include DNA, RNA, or a combination thereof. In some embodiments, a coding region, an open reading frame, or a combination thereof. In some embodiments, the CMS gene may be a maternal trait conferred by the mitochondrial genome that renders the production of functional pollen and / or male reproductive organs ineffective, except in the presence of a reproductive capacity restoration (RF) gene.In some embodiments, chimeric mitochondrial ORFs may be found to result in male sterility and produce unisex-female plants. In some embodiments, the creation of chimeric CMS genes may result in the plant mitochondrial genome being highly recombinant and repetitive. In some embodiments, the methods described herein can be used to introduce one or more naturally occurring or custom-designed CMS protein-coding sequences into the mitochondria of various monocotyledonous, dicotyledonous, or combinations thereof. In some embodiments, monocotyledonous species may include wheat, maize, rice, barley, sorghum, sugarcane, rye, or any combination thereof. In some embodiments, dicotyledonous plants may include soybeans, potatoes, tomatoes, canola, broccoli, cauliflower, or any combination thereof. In some embodiments, the CMS protein-coding sequence of the CMS gene can be manipulably ligated to heterologous regulatory sequences.

[0221] In some embodiments, the CMS gene may comprise all or part of the orf79 gene (e.g., the orf79 protein-coding sequence) derived from rice. In some embodiments, the CMS gene may have an amino acid sequence having at least about 50% sequence identity with SEQ ID NO: 47 (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the CMS gene may comprise all or part of the orf256 gene (e.g., the orf256 protein-coding sequence) derived from wheat. In some embodiments, the CMS gene may have an amino acid sequence having at least about 50% sequence identity with SEQ ID NO: 54 (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the CMS gene may comprise all or part of the wheat-derived orf279 gene (e.g., the orf279 protein-coding sequence). In some embodiments, the CMS gene may have an amino acid sequence having at least about 50% sequence identity with SEQ ID NO: 56 (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the CMS gene may comprise all or part of the maize-derived T-urf13 gene (e.g., the T-urf13 protein-coding sequence).

[0222] Selection of transformed cells using plant enzyme inhibitors In some embodiments, plant embryogenetic callus cultures can be initiated on a Chu-N6-based induction and maintenance medium supplemented with plant growth regulator 2,4-D and maintained for a minimum of 4–8 weeks (e.g., 4–6 weeks). In some embodiments, the plant may be selected from the group consisting of rice, wheat, maize, sorghum, barley, rye, canola, broccoli, cauliflower, and soybean. In some embodiments, the plant is rice. In some embodiments, callus can be prepared for transformation by seeding tissue into a target area on the same N6-based medium supplemented with mannitol and sorbitol for osmotic protection four days before transformation.

[0223] In some embodiments, plant callus (e.g., rice callus) can be transformed with one or more plant enzyme expression constructs (e.g., herbicide-resistant plant enzymes, regulatory plant enzymes). In some embodiments, plant callus can be transformed with one or more plant enzymes (e.g., herbicide-resistant ALS, herbicide-resistant EPSPS, and / or herbicide-resistant GS expression constructs). For example, the herbicide-resistant GS gene or EPSPS gene can be transformed simultaneously with the herbicide-resistant ALS gene. In some embodiments, the expression cassette for the herbicide-resistant plant enzyme is a mitochondrial expression cassette. In some embodiments, the expression cassette is a nuclear expression cassette encoding a plant enzyme (or a variant of the plant enzyme) fused to a mitochondrial target sequence.

[0224] In some embodiments, plant callus (e.g., rice callus) can be transformed with one or more ALS expression constructs (e.g., herbicide-resistant ALS-LS, regulatory ALS-SS). In some embodiments, the herbicide-resistant ALS-LS expression cassette is a mitochondrial expression cassette. In some embodiments, the ALS-SS expression cassette is a mitochondrial expression cassette. In some embodiments, the ALS-SS (or herbicide-resistant ALS-LS) expression cassette is a nuclear expression cassette encoding ALS-SS (or herbicide-resistant ALS-LS) fused with a mitochondrial targeting sequence.

[0225] In some embodiments, the herbicide-resistant ALS may comprise an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 24. In some embodiments, the polynucleotide encoding the herbicide-resistant ALS may comprise a nucleic acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 25. In some embodiments, the modified regulatory subunit of acetolactate synthase or the modified biologically active fragment may contain an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 21.

[0226] In some embodiments, plant callus (e.g., rice callus) can be transformed with one or more EPSPS expression constructs (e.g., herbicide-resistant EPSPS, regulatory EPSPS, ATP1 promoter driving the coding region of the herbicide-resistant form of EPSPS). In some embodiments, the herbicide-resistant EPSPS expression cassette is a mitochondrial expression cassette. In some embodiments, the EPSPS expression cassette is a mitochondrial expression cassette. In some embodiments, the EPSPS (or herbicide-resistant EPSPS) expression cassette is a nuclear expression cassette encoding EPSPS (or herbicide-resistant EPSPS) fused with a mitochondrial targeting sequence.

[0227] In some embodiments, the herbicide-resistant EPSPS may comprise an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 62. In some embodiments, the polynucleotide encoding the herbicide-resistant EPSPS may comprise a nucleic acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 63.

[0228] In some embodiments, plant callus (e.g., rice callus) can be transformed with one or more GS expression constructs (e.g., herbicide-resistant GS, regulatory GS, ATP1 promoter driving the coding region of the herbicide-resistant form of GS). In some embodiments, the expression cassette for herbicide-resistant GS is a mitochondrial expression cassette. In some embodiments, the GS expression cassette is a mitochondrial expression cassette. In some embodiments, the GS (or herbicide-resistant GS) expression cassette is a nuclear expression cassette encoding GS (or herbicide-resistant EPSPS) fused with a mitochondrial targeting sequence.

[0229] In some embodiments, the herbicide-resistant GS may comprise an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 72. In some embodiments, the polynucleotide encoding the herbicide-resistant GS may comprise a nucleic acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 73.

[0230] In some embodiments, the sequence encoding the start codon of a naturally occurring polypeptide variant with enzymatic activity (e.g., herbicide-resistant ALS, herbicide-resistant EPSPS, or herbicide-resistant GS) can be replaced with the sequence encoding a mitochondrial RNA editing site. For example, the mitochondrial RNA editing sites may include the rice mitochondrial nad4L gene (e.g., SEQ ID NO: 41), the rice mitochondrial cox2 gene (e.g., SEQ ID NO: 42), the wheat mitochondrial cox2 gene (e.g., SEQ ID NO: 80), and any combination thereof.

[0231] In some embodiments, transformation is performed using techniques selected from the group consisting of microinjection, meristematic transformation, electroporation, Agrobacterium-mediated transformation, virus-based gene transfer, transfection, vacuum infiltration, microparticle gun bombardment, or any combination thereof. In some embodiments, transformation may be performed using microparticle gun bombardment. In some embodiments, changes in transformation conditions may include changes in particle size and quantity. In some embodiments, changes in transformation conditions may include changes in the amount of DNA on the particles. In some embodiments, changes in transformation conditions may be the concentration of the selector in the first selection after bombardment, or in subsequent selections. In some embodiments, the following steps may be performed for culturing, selection, and regeneration.

[0232] After bombardment, the callus can be incubated in the dark at 26°C for 16–20 hours, and then the aggregates, approximately 1–3 mm in size, can be subcultured in a selective medium. In some embodiments, the selective medium is supplemented with an inhibitor of a plant enzyme (e.g., ALS).

[0233] In some embodiments, the plant enzyme may be ALS, and the inhibitor of ALS is sulfonylurea. In some embodiments, the inhibitor of ALS is chlorsulfuron. In some embodiments, the selective medium is supplemented with chlorsulfuron at concentrations of 20 nM to 100 nM, 100 nM to 1 μM, 1 μM to 20 μM, or 20 μM to 100 μM. In some embodiments, the plant enzyme may be EPSPS, and the inhibitor of EPSPS is glyphosate. In some embodiments, the selective medium may be supplemented with glyphosate at concentrations of at least about 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or more. In some embodiments, the plant enzyme may be GS, and the inhibitor of GS may be glyphosate, bialaphos, or fosalacin. In some embodiments, the selective medium may be supplemented with glufosinate at concentrations of 1–1000 mg / L, 10–500 mg / L, 20–400 mg / L, 30–300 mg / L, 40–200 mg / L, or 50–100 mg / L.

[0234] Next, the callus on the selective medium can be returned to incubation in the dark for 2-3 weeks. After 2-3 weeks of incubation in the dark, small aggregates of about 1-3 mm in size can be subcultured again in fresh selective medium containing a plant enzyme inhibitor (e.g., chlorsulfuron) and incubated for about 2 weeks in a plant growth chamber illuminated at 26°C with a light cycle of 16 hours light to 8 hours dark, an intensity of 60 μmol / m² / second. In some embodiments, further subculture in fresh selective medium can be performed after a 2-week maintenance period in light.

[0235] At the end of the second selection period or thereafter, 5–8 weeks after bombardment, the actively growing calluses (individual initial events) can be separated from the surrounding dead tissue and transferred to individual plates of fresh selective medium supplemented with a plant enzyme inhibitor (e.g., chlorsulfuron) to maintain their individual identity.

[0236] At the end of this final selection period of more than two weeks for individual plates, callus that maintains growth (representing a presumed mitochondrial transformation event) can be transferred to an N6-based medium for embryo maturation, which still contains a plant enzyme inhibitor (e.g., chlorsulfuron) as a selector, but has had the growth regulator 2,4-D removed and is supplemented with 2.5 g / L of Phytagel.

[0237] After at least 10–14 days, mature somatic embryos showing signs of normal maturation can be transferred to an N6-based germination medium still containing a plant enzyme inhibitor (e.g., chlorsulfuron) as a selective agent. In some embodiments, this medium can be supplemented with the growth regulators 0.2 mg / L naphthaleneacetic acid and 2 mg / L 6-benzylaminopurine, as well as 2.5 g / L Phytagel.

[0238] In some embodiments, these organisms can be grown in a continuous light growth environment at 26–28°C for root and shoot formation. In some embodiments, these organisms can be grown at 26–28°C in a 16-hour / 8-hour light / dark growth chamber for root and shoot formation.

[0239] In some embodiments, plants that show development of both roots and shoots after the previous step may be transferred to pots containing an artificial potting medium and gradually adapted to greenhouse conditions. The plants can grow in the greenhouse until maturation and seed production.

[0240] In some embodiments, immature plant scutellae can be used. Approximately 24 hours before transformation, immature scutellae of wheat cultivars Fielder and / or Bobwhite, approximately 2 mm in length, can be prepared for transformation by excising them from immature seeds, removing the hypocotyls, and sowing them in circular target areas on a hyperosmolar medium. In some embodiments, the medium may include agar coagulation MS basic medium supplemented with amino acids, sucrose, and 2,4-D, with or without the addition of cefotaxime antibiotic at a rate of 250 mg / L for contamination control.

[0241] In some embodiments, pre-cultured wheat blastodiscs can be transformed with one or more plant enzyme expression constructs (e.g., herbicide-resistant plant enzymes, regulatory plant enzymes) using a particle bombardment method. In some embodiments, the blastodiscs can be transformed simultaneously with mitochondrial oligomycin resistance genes (oliRs) linked to the plant enzymes described herein.

[0242] In some embodiments, immediately after bombardment, or up to two days after bombardment, the blastodisc may be spread across the entire plate that received the bombardment, or spaced apart on additional new plates of the same hyperosmolar medium and incubated in the dark at 26°C for up to seven days. In some embodiments, the cultured blastodisc may be transferred to a selective callus induction medium (e.g., an MS-based hyperosmolar medium supplemented with plant enzymes and / or cefotaxime inhibitors). In some embodiments, the culture may be incubated in the dark for at least one, two, three, four, or five weeks.

[0243] In some embodiments, after incubation on a selective callus induction medium containing a plant enzyme inhibitor, the blastodisc can be continuously maintained on an MS-based selective callus induction medium containing a plant enzyme inhibitor (e.g., chlorsulfuron). In some embodiments, after incubation on a selective callus induction medium containing a plant enzyme inhibitor, the blastodisc can be transferred to a first-stage agarose coagulation and regeneration medium (RZ) supplemented with maltose, 2,4-D, zeatin, and silver nitrate in the presence of the plant enzyme inhibitor. In some embodiments, the use of cefotaxime can be discontinued after 3–6 weeks of culture on the callus induction medium.

[0244] In some embodiments, blastodiscs on callus induction medium can be cultured under light (16 / 8 photoperiod) and transferred approximately every 3 weeks for 32 weeks to fresh medium containing a plant enzyme inhibitor (e.g., chlorsulfuron). In some embodiments, callus induced from individual bombardmented blastodiscs can be subdivided into smaller pieces while maintaining their original identity.

[0245] In some embodiments, the embryodisk on the shoot-inducing medium can be subcultured every three weeks in fresh first-stage regeneration medium and cultured under light until shoot formation can be confirmed. In some embodiments, selected green portions of the callus and small shoots can be transferred to a second-stage regeneration medium (RO) which is the same as the first-stage regeneration medium but without growth regulators. The developing plants can be transferred to a dome-shaped transparent culture container and grown to a planting size. In some embodiments, the developing plants can be planted in soil and acclimatized in a greenhouse.

[0246] Double selection process In some embodiments, the method described herein may have multiple selection processes. In some embodiments, a gene expression cassette containing a plant enzyme or a variant thereof (e.g., herbicide-resistant ALS) can be transformed simultaneously with a second selectable marker expression cassette, e.g., a 35S:HPT nuclear expression cassette conferring hygromycin B resistance. In some embodiments, the selection medium may comprise one or more inhibitors of the plant enzyme and a selector (e.g., hygromycin B). In some embodiments, the selection medium may comprise one or more inhibitors of the plant enzyme (e.g., chlorsulfuron) and at least about 1 to 100 mg / L, 10 to 75 mg / L, or 25 to 50 mg / L of hygromycin B.

[0247] In some embodiments, a gene expression cassette containing a plant enzyme or its variant (e.g., herbicide-resistant ALS) can be linked to an oliR expression cassette that confers resistance to the antibiotic oligomycin, and transformation can occur simultaneously. In some embodiments, oligomycin can be incorporated into the selective medium at concentrations of approximately 0.5–100 mg / L, 1–50 mg / L, 1–50 mg / L, or 1–5 mg / L. In some embodiments, the carbon source (e.g., sucrose) may be reduced to approximately 0.1% (1 mg / L), 0.2% (2 mg / L), 0.3% (3 mg / L), 0.4% (4 mg / L), 0.5% (5 mg / L), 0.6% (6 mg / L), 0.7% (7 mg / L), 0.8% (8 mg / L), or 0.9% (9 mg / L) per liter of selective medium, or it may be replaced with at least approximately 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL of sterile 50% glycerol solution per liter of selective medium.

[0248] In some embodiments, the compound disulfiram can also be incorporated into the selective medium at concentrations of approximately 20 μM, 40 μM, 60 μM, 800 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, or 500 μM to inhibit the cells' ability to utilize any alcohol produced by anaerobic respiration of the treated cells. In some embodiments, the gene expression cassette containing a plant enzyme or a variant thereof (e.g., a herbicide-resistant ALS gene) can be composed of a geminivirus VOR sequence.

[0249] In some embodiments, a gene expression cassette containing a plant enzyme or a variant thereof (e.g., herbicide-resistant ALS) can be simultaneously transformed with a second selectable marker expression cassette containing a polynucleotide encoding a phosphyte dehydrogenase enzyme or a biologically active fragment thereof. In some embodiments, the selective medium may contain one or more inhibitors of the plant enzyme and a selector (e.g., a phosphyte). In some embodiments, the selective medium may contain one or more inhibitors of plant enzymes (e.g., chlorsulfuron), as well as at least about 0.1–0.25 mM, 0.25–0.5 mM, 0.5–0.75 mM, 0.75–1.0 mM, 1.0–2.5 mM, 2.5–5.0 mM, 5.0–7.5 mM, 7.5–10 mM, 10–15 mM, 15–20 mM, 20–25 mM, 25–30 mM, 30–35 mM, 35–40 mM, 40–45 mM, and 45–50 mM.

[0250] In some embodiments, a gene expression cassette containing a plant enzyme or a variant thereof (e.g., a herbicide-resistant ALS gene) can be simultaneously transformed with at least one expression cassette conferring resistance to an additional selective marker (e.g., hygromycin B resistance or oligomycin resistance), at least two expression cassettes (e.g., conferring hygromycin B resistance and oligomycin resistance), or at least three expression cassettes (e.g., conferring hygromycin B resistance, oligomycin resistance, and further selective agents). In some embodiments, one or more selective agents (e.g., hygromycin B) can be added instead of or in addition to a plant enzyme inhibitor. In some embodiments, the timing of initiation of one or more selective agents in combination with a plant enzyme inhibitor (e.g., chlorsulfuron) can be varied. For example, after one or more cycles of initial subculturing and selection, the use of the plant enzyme can be interrupted, and one or more selective agents (e.g., oligomycin) can be utilized.

[0251] Inducible expression system In some embodiments, the target tissue for plant (e.g., rice) transformation (e.g., particulate gun transformation) may be derived from different sources. Different sources of callus tissue may be derived from the aforementioned Agrobacterium tumefaciens transformation. In some embodiments, a dexamethasone-inducible system can be used to produce the Geminivirus Rep protein. In some embodiments, to supply sufficient tissue for bombardment, this event can be maintained on a first selective medium supplemented with 40 mg / L hygromycin, amino acids, proline, maltose, and 2,4-D growth regulators prior to transformation.

[0252] In some embodiments, the tissue to be bombarded may be derived from an induction line pre-cultured for 4 days prior to bombardment on a first selective medium. In some embodiments, the pre-culture medium may be supplemented with 1,000 μl of 10 μM dexamethasone (DEX). In some embodiments, DMSO may be used as a control.

[0253] In some embodiments, callus can be prepared for bombardment by seeding tissue into a target area on an N6-based callus induction medium that is supplemented with mannitol and sorbitol for osmotic protection but does not contain DEX. After bombardment, the callus can be incubated in the dark at 26°C for 16–20 hours, and then the callus tissue aggregates, approximately 1–3 mm in size, can be subcultured in an N6-based callus maintenance medium supplemented with the growth regulator 2,4-D, a suitable inhibitor of plant enzymes (e.g., 20–100 nM chlorsulfuron), and a suitable selector containing one or more additional selective markers (e.g., 25–50 mg / L hygromycin and 1–5 mg / L oligomycin), with the carbon source reduced or replaced. In some embodiments, chemical induction with DEX in the medium can be initiated in the initial selection. The callus on the selective medium with DEX can then be returned to incubation in the dark for the initial selection. In some embodiments, DEX was introduced at a later point in time after the selection process. After incubation in the dark for 2-3 weeks, small (1-3 mm) aggregates can be subcultured again in fresh selective medium and then incubated as described. After maintaining in light for 2-3 weeks, further subculture in fresh selective medium containing DEX and / or DMSO may be optimal. In some embodiments, induction with DEX can be continued throughout the selection process. In some embodiments, the use of DEX and / or DMSO can be discontinued for one or more culture periods. In some experiments, DEX can be reintroduced at a point later in the selection process.

[0254] Screenable and selectable markers In some embodiments, polynucleotides (e.g., donor polynucleotides) may also encode phenotypic markers. In some embodiments, phenotypic markers may be screenable or selectable markers, which may include visually screenable markers, selectable markers, or combinations thereof. In some embodiments, selectable markers may include positive or negative selectable markers. In some embodiments, any phenotypic marker may be used. In some embodiments, selectable or screenable markers may include DNA segments, and DAN segments may enable the identification or selection of molecules or cells containing them, for example, under certain conditions. In some embodiments, markers may encode activities such as, but not limited to, the production of RNA, peptides, or proteins, or may provide a binding site to RNA, peptides, proteins, inorganic and organic compounds, or compositions, etc.

[0255] In some embodiments, examples of selectable or screenable markers include, but are not limited to, DNA segments containing restriction enzyme sites, DNA segments encoding products that confer resistance to other toxic compounds including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, and hygromycin, DNA segments encoding products lacking in recipient cells (e.g., tRNA genes, nutrient requirement markers), DNA segments encoding easily identifiable products (e.g., phenotypic markers such as β-galactosidase and GUS, fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cell surface proteins), generation of novel primer sites for PCR (e.g., juxtaposition of two previously unjuxtaposed DNA sequences), inclusion of DNA sequences that have not been or have been acted upon by restriction endonucleases or other DNA modifying enzymes, chemicals, etc., and inclusion of DNA sequences required for specific modifications (e.g., methylation) that enable identification, or any combination thereof.

[0256] In some embodiments, further selectable markers may include polynucleotides encoding proteins that can confer resistance to herbicides such as glyphosate, sulfonylurea, glufosinate ammonium, bromoxynyl, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). In some embodiments, herbicide-resistant proteins may include herbicide-resistant forms of the following: acetyl coenzyme A carboxylase (ACCase), 4-hydroxyphenylpyrubate dioxygenase (HPPD), sulfonylurea-resistant acetolactic acid synthase (ALS), imidazolinone-resistant acetolactic acid synthase (ALS), glyphosate-resistant 5-enolpyruvirschimate-3-phosphate synthase (EPSPS), glyphosate-resistant glyphosate oxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), phosphinotricin acetyltransferase (PAT), protoporphyrinogen oxidase (PPO or PROTOX), auxin enzymes or receptors, P450 polypeptides, or any combination thereof. In some embodiments, genes encoding the above proteins can be cited as non-limiting examples of genes useful for conferring herbicide resistance to plants. In some embodiments, the neomycin phosphotransferase II (nptII) gene can encode a protein that confers resistance to the antibiotics kanamycin and geneticin, and the hygromycin phosphotransferase (HPT) gene can encode a protein that confers resistance to hygromycin.

[0257] In some embodiments, DNA transformation of organelle genomes can be carried out, for example, in plastids and mitochondria. In some embodiments, selectable marker genes may include, for example, photosynthesis (atpB, tscA, psaA / B, petB, petA, ycf3, rpoA, rbcL), antibiotic resistance (rrnS, rrnL, aadA, nptII, aphA-6), herbicide resistance (psbA, bar, AHAS(ALS), EPSPS, HPPD, sul), and metabolism (BADH, codA, ARG8, ASA2) genes. In some embodiments, the bacterial-derived sul gene may contain dihydropteroate synthase activity that is insensitive to the herbicide sulfonamide and can be used as a selectable marker when the protein product is targeted to plant mitochondria.

[0258] In some embodiments, a sequence encoding a marker can be incorporated into the organelle genome. In some embodiments, the incorporated sequence encoding the marker can then be removed from the transformed organelle genome. In some embodiments, removal of the marker-encoding sequence can be facilitated by the presence of direct repeats before and after the region encoding the marker. In some embodiments, removal of the marker-encoding sequence can be performed by the organelle's endogenous homologous recombination system or by the use of a site-directed recombinase system such as cre-lox or site-directed recombination. In some embodiments, site-directed recombination may include FLP-FRT recombination.

[0259] In some embodiments, caspase-activatable GFP (CA-GFP) is a modified form of GFP in which fluorescence is completely quenched by the association of a hydrophobic quenching peptide that tetramerizes GFP and prevents chromophore maturation. In some embodiments, the sequence of the CA-GFP protein may correspond to GFP having a fusion of DEVDFQGPCNDSSDPLVVAASIIGILHLILWILDRL (SEQ ID NO: 2) at the carboxy terminus. In some embodiments, a caspase recognition sequence (SEQ ID NO: 3) containing the amino acid DEVD may be present in CA-GFP between the fluorescence domain and the quenching domain. In some embodiments, GFP fluorescence can be completely restored in vivo by catalytic removal of the quenching peptide by caspase-mediated cleavage. In some embodiments, the nucleic acid sequence encoding CA-GFP can be modified by replacing the caspase recognition sequence with a mitochondrial RNA editing sequence. In some embodiments, the RNA editing sequence can be selected such that the C-to-U conversion creates a stop codon in the mRNA. In some embodiments, expression of nucleic acid sequences encoding modified CA-GFP provides a screenable marker because it causes quenching in the cytoplasm or plastids but fluorescence in mitochondria. In some embodiments, candidate RNA editing sequences for this purpose are located in the wheat mitochondrial cox2 gene at positions 449, 587, and 620. In some embodiments, candidate RNA editing sequences for this purpose located in the wheat mitochondrial cox2 gene at positions 449, 587, and 620 may include SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs: 6, respectively.

[0260] In some embodiments of this specification, methods are disclosed that can provide a transformation efficiency to organelles (e.g., mitochondria, plastids) of at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0261] Use of multiple selectable and / or screenable markers The systems and methods described herein may utilize at least one, at least two, at least three, at least four, or at least five selectable or screenable markers. Selectable marker genes commonly used in plants may include, for example, genes that confer resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aadA) and gentamicin (aac3 and aacC4), or genes that confer resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO), and glyphosate (aroA or EPSPS). In some cases, screenable markers may provide the ability to visually screen transformants such as those expressing luciferase or green fluorescent protein (GFP), or uidA genes (GUS) or β-glucuronidase known to various chromogenic substrates. In some embodiments, one or more selectable or screenable markers may be used at different developmental stages in cells, tissues, reproductive materials, seeds, pollen, offspring, or any combination thereof. For example, cells may be transformed simultaneously with a first selectable marker (e.g., a gene conferring resistance to the antibiotic hygromycin) and a second selectable marker (herbicide-resistant ALS-LS), and may grow at different developmental stages in the presence of the first selectable agent (hygromycin) and then the second selectable agent (e.g., an ALS inhibitor). Transformation may also occur in a non-selective manner during one or more stages or processes of development or regeneration of the transformed cells, tissues, reproductive materials, seeds, pollen, offspring, or any combination thereof. In some embodiments, one or more selectable or screenable markers may be incorporated into various organelles (e.g., nuclear genome and mitochondrial genome). In some embodiments, one or more selectable or screenable markers may be removed upon successful transformation.

[0262] Herbicide-resistant ALS large subunit as a selectable marker The acetolactate synthase large subunit (ALS-LS; EC:2.2.1.6) catalyzes the first common step in the biosynthetic pathways of the branched-chain amino acids leucine, isoleucine, and valine. Acetolactic acid synthase activity involves converting two molecules of piruvate into one molecule of acetolactate and one molecule of carbon dioxide. Acetolactic acid synthase is also known as acetohydroxy acid synthase (AHAS). In some cases, acetolactate synthase also has a regulatory small subunit (ALS-SS). In some cases, the regulatory small subunit stimulates the activity of the acetolactate synthase catalytic large subunit 7-10 times and confers sensitivity to inhibition by valine and activation by ATP. In yeast, ALS is located in the mitochondria. In yeast, the genes for the large and small subunits are encoded in the nucleus, and the primary translation product has a mitochondrial target sequence. In plants and algae, ALS is located in the plastids. In plants and algae, the genes for the large and small subunits of ALS are encoded in the nucleus, and the primary translation product contains a plastid target sequence.

[0263] In some cases, ALS inhibitors are used as herbicides by inhibiting the production of branched-chain amino acids. These inhibitors are not a chemical class, but rather a mechanistic class with diverse chemical properties. The ALS inhibitor family includes sulfonylurea (SU), imidazolinone (IMI), triazolopyrimidine (TP), pyrimidinyl benzoate (PYB), sulfonanilide, and sulfonylaminocarbonyltriazolinone (SCT). ALS herbicides do not bind to the catalytic site, but instead bind to a site specific to herbicidal activity. Consequently, resistance mutations exhibit a wide-ranging effect relative to normal ALS catalytic activity, i.e., positive, negative, and neutral. For example, resistance in corngrass (Hordeum murinum) by a proline-to-serine substitution at amino acid 197 has been found to increase ALS activity by 2-3 times. Herbicide resistance to ALS has been identified in many weed species.

[0264] In some embodiments, herbicide-resistant ALS-LS may include modified Km, modified Vmax, modified cofactor affinity, modified cofactor specificity, modified thermal stability, modified feedback regulation, or any combination thereof.

[0265] In some embodiments, herbicide-resistant ALS-LS can be identified from herbicide-resistant weed populations. In some embodiments, herbicide-resistant weed populations may originate from the following species: Xanthium strumarium, Kochia scoparia, Amaranthus hybridus, Apera spica-venti, Amaranthus powellii, and Oryza sativa var. sylvantica. In some embodiments, herbicide-resistant ALS-LS may have at least one mutation in any of the following amino acids: Ala-122, Pro-197, Ala-205, Asp-376, Arg-377, Trp-574, Ser-653, Gly-654, and any combination thereof. In some cases, the number of amino acids is standardized relative to the Arabidopsis thaliana sequence.

[0266] In some embodiments, herbicide-resistant ALS-LS can be intentionally selected, for example, by laboratory selection. In some embodiments, the herbicide-resistant population may originate from the following species: Oryza sativa, Zea mays, Arabidopsis thaliana, Camelina sativa, and Sorghum bicolor. In some embodiments, the herbicide-resistant ALS-LS may be derived from Oryza sativa and may have at least one of the following amino acid mutations: Trp548Leu, Ser627Ile, Trp548Met, Ser627Asn, and any combination thereof. In some embodiments, the herbicide-resistant ALS-LS may be derived from Zea mays and may have at least one of the following amino acid mutations: Pro165Ser, Pro165Ala, Pro165Leu, and Pro165Trp. In some embodiments, herbicide-resistant ALS-LS may be derived from Arabidopsis sariana and may have at least one of the following amino acid mutations: Ser653Asn, Ala122Thr, Ala122Val, Ala205Val, Trp574Ser, Trp574Leu, Ser653Asn, Pro197Ala, and any combination thereof. In some embodiments, herbicide-resistant ALS-LS may be derived from Camelina sativa and may have at least one of the following amino acid mutations: Ala122Thr, Pro197Ser, Trp574Leu, and any combination thereof. In some embodiments, herbicide-resistant ALS-LS may be derived from Sorghum bicolor and may have at least one of the following amino acid mutations: Val531Ile, Trp545Leu, and any combination thereof.

[0267] In some embodiments, herbicide-resistant ALS-LS may be of plant origin. In some embodiments, herbicide-resistant ALS-LS may lack a chloroplast transit sequence. In some embodiments, a polynucleotide encoding herbicide-resistant ALS-LS lacking a chloroplast transit sequence can be introduced into mitochondria. In some embodiments, the enzyme may contain both herbicide-resistant ALS-LS and regulatory ALS-SS. In some embodiments, ALS-SS may be of plant origin. In some embodiments, ALS-SS may lack a chloroplast transit sequence. In some embodiments, ALS-SS is fused to a mitochondrial targeting sequence. In some embodiments, a polynucleotide encoding ALS-SS fused to a mitochondrial targeting sequence can be introduced into the nuclear genome. In some embodiments, a polynucleotide encoding ALS-SS lacking a chloroplast transit sequence can be introduced into mitochondria. In some embodiments, herbicide-resistant ALS-LS may be derived from Oryza sativa. In some embodiments, ALS-SS may be derived from Oryza sativa. In some embodiments, the presence of herbicide-resistant ALS-LS in mitochondria may enable intracellular synthesis of branched-chain amino acids (valine, leucine, and isoleucine) in the presence of an acetolactate synthase inhibitor, which may allow its use as a selectable marker.

[0268] Benefits of organisms having mitochondria transformed to express polypeptides with herbicide-resistant enzyme activity In some embodiments, introducing a polynucleotide encoding a polypeptide with herbicide-resistant enzyme activity into mitochondria may allow selection of plant or algal cells with stably transformed mitochondria. In some embodiments, transforming mitochondria with a polynucleotide encoding a polypeptide with herbicide-resistant enzyme activity may allow simultaneous transformation with additional polynucleotides of interest. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 100% of the mitochondrial genome in a cell may be transformed. In some embodiments, the cell may have isocytoplasm with respect to the transformed mitochondria.

[0269] In some embodiments, the methods described herein can promote the growth or cultivation of a target plant containing an edited mitochondrial genome while suppressing the growth of an undesirable plant (e.g., a weed) that does not contain an edited mitochondrial genome. For example, a group of plants may grow in the presence of an inhibitor of a plant enzyme described herein (e.g., ALS, EPSPS, GS), and at least one of the group of plants of choice includes mitochondria having a polypeptide having herbicide-resistant plant enzyme activity or a heterologous polynucleotide encoding a bioactive fragment thereof, and at least one of the group of plants of choice (e.g., a weed) lacking mitochondria having a polypeptide having herbicide-resistant acetolactate synthase activity or a heterologous polynucleotide encoding a bioactive fragment thereof. In some embodiments, the presence of a plant enzyme inhibitor is sufficient to selectively promote the growth of at least one of the group of plants of choice, resulting in increased growth of at least one of the group of plants of choice compared to an undesirable plant (e.g., a weed) lacking the polypeptide having herbicide-resistant acetolactate synthase activity or a bioactive fragment thereof. In some embodiments, plant enzyme inhibitors may be applied to plants, multiple plants, soil adjacent to plants, or any combination thereof. In some embodiments, plant enzyme inhibitors may be applied as leaf conditioners, soil conditioners, or any combination thereof. In some embodiments, plant enzyme inhibitors may be soluble in water and applied to plants, multiple plants, soil adjacent to plants, or any combination thereof.

[0270] In some embodiments, plants having mitochondria transformed with polynucleotides encoding polypeptides possessing herbicide-resistant plant enzyme activity can transmit the transformed mitochondria to offspring plants through maternal inheritance. In some embodiments, plants having mitochondria transformed with polynucleotides encoding polypeptides possessing herbicide-resistant plant enzyme activity may exhibit less horizontal gene transfer (e.g., to weed species) than plants having nuclear genomes transformed with polynucleotides encoding polypeptides possessing herbicide-resistant acetolactate synthase activity.

[0271] Methods utilizing a two-component RNA guide and a polynucleotide-derived polypeptide system In some embodiments, the polynucleotide-derived polypeptide systems described herein may be particularly useful for genomic manipulation in situations where endonuclease off-target cleavage may be toxic to target cells. In some embodiments, the polynucleotide-derived polypeptide systems described herein, the constant component, and the polynucleotide encoding the organelle-targeted polynucleotide-derived polypeptide can be stably incorporated into the nuclear genome of a cell. In some embodiments, the polynucleotide encoding the organelle-targeted polynucleotide-derived polypeptide can be transiently expressed in the nuclear genome of a cell. In some embodiments, the polynucleotide can encode a modified polynucleotide-derived polypeptide, including an enzymatically active polynucleotide-derived polypeptide (e.g., Cas polypeptide, MAD polypeptide) fused to an organelle transport sequence (e.g., a mitochondrial-targeted peptide or a chloroplast-targeted peptide). In some embodiments, the expression of the polynucleotide encoding the modified polynucleotide-derived polypeptide may be under the control of a promoter. In some embodiments, the promoter may be a constitutive promoter, a tissue-specific promoter, or an inductive promoter, such as a temperature-inducible, stress-inducible, developmental-stage-inducible, or chemically inducible promoter. In some cases, in the absence of a variable component (e.g., guide RNA or crRNA), polynucleotide-derived polypeptides may not cleave target nucleic acids. In the absence of a variable component (e.g., guide RNA or crRNA), the presence of polynucleotide-derived polypeptides in cells (e.g., plant cells) may have little or no effect. In some embodiments, polynucleotide-derived polypeptide systems can be used to create and / or maintain cell lines or transgenic organisms capable of efficient expression of polynucleotide-derived polypeptides. Expression of polynucleotide-derived polypeptides in cell lines or transgenic organisms may have little or no effect on cell viability.

[0272] In some embodiments, guide polynucleotides (e.g., guide RNA or crRNA) can be introduced by various means into cells containing an expression cassette stably incorporated and expressed to a polynucleotide-derived polypeptide in order to induce cleavage at a desired genomic site to achieve modification of a target gene. In some embodiments, guide polynucleotides (e.g., guide RNA or crRNA) can be synthesized chemically or enzymatically and introduced into polynucleotide-derived polypeptide-expressing cells expressing cells by direct delivery methods such as particle bombardment or electroporation. In some embodiments, the guide polynucleotide can be fused to an RNA molecule that enables transport to an organelle. In some embodiments, the guide polynucleotide can be fused to an RNA molecule that enables binding to a protein that facilitates transport to an organelle. In some embodiments, the guide polynucleotide can be transported to an organelle by association with a modified polynucleotide-derived polypeptide containing an enzymatically active polynucleotide-derived polypeptide fused to an organelle transport sequence.

[0273] In some embodiments, the gene can efficiently express a guide polynucleotide in target cells. In some embodiments, the guide polynucleotide may include guide RNA, crRNA, or a combination thereof. In some embodiments, the gene capable of efficiently expressing a guide polynucleotide in target cells may be synthesized chemically, enzymatically, or in a biological system. In some embodiments, the gene capable of efficiently expressing a guide polynucleotide in target cells may be introduced into polynucleotide-derived polypeptide-expressing cells via direct delivery, biological delivery, or a combination thereof. In some embodiments, direct delivery may include particle bombardment, electroporation, vacuum infiltration, or any combination thereof. In some embodiments, biological delivery may include Agrobacterium-mediated DNA delivery.

[0274] In some embodiments, a method for modifying the genome of an organelle may include the step of introducing a first polynucleotide encoding at least one guide polynucleic acid into the organelle. In some embodiments, the at least one guide polynucleic acid can induce a polynucleotide guide polypeptide to cleave at least one target sequence present in the organelle genome. In some embodiments, the guide polynucleic acid may include a guide RNA. In some embodiments, the polynucleotide-derived polypeptide may include a Cas polypeptide, a Cas9 polypeptide, or a combination thereof. In some embodiments, the method may further include the step of introducing a second polynucleotide into the organelle. In some embodiments, the second polynucleotide may encode a polynucleotide-derived polypeptide. In some embodiments, the polynucleotide-derived polypeptide, upon association with the guide polynucleic acid, can cleave at least one target sequence. In some embodiments, the method may further include the step of introducing a third polynucleotide encoding at least one homologous organelle DNA sequence into the organelle. In some embodiments, the at least one homologous organelle DNA may be of sufficient size for homologous recombination. In some embodiments, at least one target sequence can be removed by incorporating at least one homologous organelle DNA sequence into the organelle DAN. In some embodiments, the organelle may include mitochondria, plastids, or a combination thereof.

[0275] In some embodiments of this specification, methods for selecting plants containing modified organelle genomes are disclosed. In some embodiments, the methods can be used to identify cells having modified genomes at or near a target site without using screenable or selectable marker phenotypes. In some embodiments, methods including, but not limited to, PCR, sequencing, nuclease digestion, Southern blotting, and any combination thereof, may include the step of directly analyzing the target sequence to detect any changes in the target sequence.

[0276] In some embodiments, sufficient homology or sequence identity may indicate that two polynucleotide sequences may have sufficient structural similarity to act as substrates for homologous recombination. In some embodiments, structural similarity may include the full length of each polynucleotide fragment, the sequence similarity of each polynucleotide, or a combination thereof. In some embodiments, sequence similarity may be described by the percentage of sequence identity over the full length of multiple sequences, by a conserved region including local similarity such as a continuous nucleotide with 100% sequence identity, by the percentage of sequence identity over a portion of the length of multiple sequences, or any combination thereof.

[0277] In some embodiments, the amount of homology or sequence identity shared by the target and donor polynucleotides may vary. For example, the length of the sequence homology may be at least about 20 bp, at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 250 bp, at least about 300 bp, at least about 400 bp, at least about 500 bp, at least about 600 bp, at least about 700 bp, at least about 800 bp, at least about 900 bp, at least about 1000 bp, at least about 1250 bp, at least about 1500 bp, at least about 1750 bp, at least about 2000 bp, at least about 2.5 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, or at least about 10 kb. In some embodiments, the amount of homology can also be described by the sequence identity percentage across the total alignment length of two polynucleotides, which may include sequence identity percentages of at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, sufficient homology may include polynucleotide length, overall sequence identity percentage, conserved regions of consecutive nucleotides, local sequence identity percentage, or any combination thereof. In some embodiments, sufficient homology may be described as a 75–150 bp region having at least 80% sequence identity with respect to the region of the target locus. In some embodiments, sufficient homology can also be described by the ability of two polynucleotides to be predicted to specifically hybridize under highly strict conditions.

[0278] In some embodiments, a plant cell having the introduced array can be grown or regenerated into a plant. In some embodiments, the plant can then be grown and pollinated with the same transformant or a different transformant or non-transformant, resulting in progeny having the desired characteristics and / or containing the introduced polynucleotide or polypeptide being identified. In some embodiments, the polynucleotide can be stably maintained and inherited, and more than two generations can be grown to ensure that seeds are harvested.

[0279] In some embodiments, any plant may be used. In some embodiments, the plant may include monocots or dicots. In some embodiments, monocots include maize (Gee maize), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), prosomillet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), maize, wheat (Triticum aestivum), and sugarcane (Saccharum). spp.)), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palm, ornamental plants, lawn, other grasses, or any combination thereof. In some embodiments, dicotyledonous plants may include soybeans (Glycine max), canola (Brassica napus and B. campestris), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis sariana), sunflower (Helianthus anus) This may include annuus, cotton (Gossypium arboreum), peanuts (Arachis hypogaea), tomatoes (Solanum lycopersicum), potatoes (Solanum tuberosum), or any combination thereof.

[0280] In some embodiments, after introducing designed changes into the organellar DNA, the next step can be to maintain the edited organellar DNA in a pool of unmodified organellar DNA and shift the balance between organellar DNAs to advantageously act on the maintenance of the genome-edited organellar DNA. In some embodiments, this can be achieved by reducing the amplification of unmodified organellar DNA. In some embodiments, the guide polynucleic acid can be designed for multiple target sites in the unmodified organellar genome. In some embodiments, the donor polynucleotide can include donor DNA. In some embodiments, the donor polynucleotide can be designed such that the target site is modified so that it is no longer recognized by the relevant polynucleotide-derivatized polypeptide system. In some embodiments, the expression of the polynucleotide-derivatized polypeptide can result in the introduction of single-stranded or double-stranded breaks into the unmodified organellar DNA, thereby increasing the proportion of the modified genome. In some embodiments, cells can be pre-treated with the relevant polynucleotide-derivatized polypeptide system to introduce breaks into the organellar DNA. In some embodiments, the pre-treatment can reduce the number of organellar DNA molecules available for homologous recombination.

[0281] In some embodiments, cells with isocytoplasmic organelle genomes may be selected. In some embodiments, cells containing multiple mitochondrial genomes may be selected, where at least 10% to 100% of the multiple mitochondrial genomes are edited. In some embodiments, selected cells may contain multiple mitochondrial genomes, of which approximately 10% to approximately 20%, approximately 10% to approximately 30%, approximately 10% to approximately 40%, approximately 10% to approximately 50%, approximately 10% to approximately 60%, approximately 10% to approximately 70%, approximately 10% to approximately 80%, approximately 10% to approximately 90%, approximately 10% to approximately 100%, approximately 20% to approximately 30%, approximately 20% to approximately 40%, approximately 20% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 70%, approximately 20% to approximately 80%, approximately 20% to approximately 90%, approximately 20% to approximately 100%, approximately 30% to approximately 40%, approximately 30% to approximately 50%, approximately 30% to approximately 60%, approximately 30% to approximately 70%, and approximately 30% to approximately 80%. Approximately 30% to 90%, approximately 30% to 100%, approximately 40% to 50%, approximately 40% to 60%, approximately 40% to 70%, approximately 40% to 80%, approximately 40% to 90%, approximately 40% to 100%, approximately 50% to 60%, approximately 50% to 70%, approximately 50% to 80%, approximately 50% to 90%, approximately 50% to 10 0%, approximately 60% to 70%, approximately 60% to 80%, approximately 60% to 90%, approximately 60% to 100%, approximately 70% to 80%, approximately 70% to 90%, approximately 70% to 100%, approximately 80% to 90%, approximately 80% to 100%, or approximately 90% to 100% contain edited mitochondrial genomes. In some embodiments, selected cells may contain multiple mitochondrial genomes, of which approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100% contain edited mitochondrial genomes. In some embodiments, the selected cells may contain multiple mitochondrial genomes, of which at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% contain edited mitochondrial genomes. In some embodiments, the selected cells may contain multiple mitochondrial genomes, of which up to about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% contain edited mitochondrial genomes.In some embodiments, the organelle may include a nucleus, mitochondria, plastids, or a combination thereof.

[0282] In some embodiments, the method may involve the use of a single guide RNA (sgRNA). In some embodiments, the variable targeting domain may be fused to a polynucleotide containing a tracrRNA sequence. In some embodiments, the method may involve the use of a double-stranded guide RNA. In some embodiments, the variable targeting domain and the tracrRNA sequence may reside on separate RNA molecules. In some embodiments, the terms “double-stranded guide RNA” and “dual guide RNA” may be used interchangeably.

[0283] In some embodiments, the expression levels of proteins, RNAs, or combinations thereof may be higher when transformed into plastids or mitochondria compared to the expression levels in the nucleus. In some embodiments, protein and / or RNA expression levels may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher in plastid or mitochondrial DNA transformation compared to nuclear DNA transformation. In some embodiments, the expression stability of proteins, transcripts, or combinations thereof may be higher in plastid or mitochondrial transformation compared to nuclear transformation.

[0284] Methods for delivery In some embodiments, any suitable delivery method can be used to introduce the compositions and molecules disclosed herein into host cells or organelles. In some embodiments, the organelles may include mitochondria, plastids, or a combination thereof. In some embodiments, the host cells may include yeast cells, plant cells, or a combination thereof. In some embodiments, the compositions may include Cas proteins, polynucleotide-derived polypeptides, guide polynucleotides, donor polynucleotides, nucleic acids encoding the compositions, or any combination thereof. In some embodiments, the compositions may be delivered simultaneously or at different times. In some embodiments, the choice of gene modification method may depend on the type of cell being transformed, the circumstances under which the transformation is occurring, or a combination thereof. In some embodiments, the circumstances under which the transformation is occurring may be in vitro, ex vivo, in vivo, in a plant, or any combination thereof.

[0285] In some embodiments, delivery methods or transformations may include viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun techniques, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, lipid nanoparticles, lipid-based vectors, polymer vectors, polyethyleneimine, poly(L-lysine), vacuum infiltration, or any combination thereof.

[0286] In some embodiments, DNA transformation may include yeast nuclear genome transformation. In some embodiments, DNA transformation can be facilitated by the development of a shuttle vector that can replicate as an autonomous plasmid in E. coli and yeast. In some embodiments, the vector system may include a low copy number plasmid and integrated DNA by homologous recombination.

[0287] In some embodiments, this specification discloses methods comprising the step of delivering the polynucleotides described herein, the vectors described herein, their transcripts, proteins translated therefrom, or any combination thereof, to a host cell or organelle. In some embodiments, this specification discloses cells produced by the methods disclosed herein, organisms produced by the methods disclosed herein, organelles containing or produced therefrom the cells disclosed herein, or any combination thereof. In some embodiments, organisms may include animals, plants, fungi, or combinations thereof. In some embodiments, polynucleotide-derived polypeptides can be delivered to cells or organelles in combination with a guide sequence, and optionally by complexing with a guide sequence.

[0288] In some embodiments, the method for introducing nucleic acids may include virus-based gene transfer methods, non-virus-based gene transfer methods, or combinations thereof. In some embodiments, the method can be used to administer nucleic acids encoding the compositions of the Disclosure to cells in a culture or host organism. In some embodiments, non-viral vector delivery systems may include DNA plasmids, RNA, naked nucleic acids, nucleic acids complexed with a delivery vehicle, or any combination thereof. In some embodiments, the delivery vehicle may include liposomes. In some embodiments, RNA may include transcripts of the vectors described herein. In some embodiments, viral vector delivery systems may include DNA viruses, RNA viruses, or combinations thereof. In some embodiments, viral vector delivery systems may have either an episomal genome or an integrated genome after delivery to cells. In some embodiments, viral vector-based systems for gene transfer may include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, or any combination thereof.

[0289] In some embodiments, adenovirus-based systems can be used. In some embodiments, adenovirus-based systems can result in transient expression of the transgene. In some embodiments, adenovirus-based vectors may have high transduction efficiency in cells and may not require cell division. In some embodiments, high titers, high expression levels, or a combination thereof can be obtained using adenovirus-based vectors. In some embodiments, adeno-associated virus ("AAV") vectors can be used to transduce target nucleic acids into cells. In some embodiments, vectors can be used to transduce target nucleic acids into cells for in vitro production of nucleic acids and peptides, for in vivo and ex vivo gene therapy procedures, or any combination thereof.

[0290] In some embodiments, cells transfected with one or more vectors described herein can be used to establish novel cell lines containing one or more vector-derived sequences. In some embodiments, cells can be transiently transfected with compositions disclosed herein. In some embodiments, transient transfection may include transient transfection with one or more vectors, transfection with RNA, or a combination thereof. In some embodiments, transiently transfected cells can be modified via the activity of the CRISPR complex. In some embodiments, cells modified by the activity of the CRISPR complex can be used to establish novel cell lines containing cells that include the modification but lack any other heterologous sequences.

[0291] In some embodiments, the compositions disclosed herein may be provided as RNA. In some embodiments, the compositions disclosed herein may be produced by direct chemical synthesis or transcribed in vitro from DNA. In some embodiments, the compositions disclosed herein may be synthesized in vitro using RNA polymerase enzymes. In some embodiments, the RNA polymerase enzymes may include T7 polymerase, T3 polymerase, SP6 polymerase, or any combination thereof. In some embodiments, the RNA may be in direct contact with the target polynucleic acid. In some embodiments, the target polynucleic acid may include target DNA. In some embodiments, the target polynucleic acid may be introduced into cells using any suitable technique for introducing nucleic acids into cells. In some embodiments, suitable techniques for introducing nucleic acids into cells may include microinjection, electroporation, transfection, or any combination thereof.

[0292] In some embodiments, the polynucleotide encoding the guide nucleic acid may include DNA or RNA. In some embodiments, the polynucleotide encoding the polynucleotide-derived polypeptide may include DNA, RNA, or a combination thereof. In some embodiments, the polynucleotide encoding the guide nucleic acid and the polynucleotide-derived polypeptide can be delivered to cells using a suitable transfection technique. In some embodiments, the nucleic acid encoding the composition of the Disclosure may be delivered on a vector or cassette. In some embodiments, the vector or cassette may include a DNA vector. In some embodiments, the vector may include a plasmid, cosmid, minicircle, phage, virus, or any combination thereof. In some embodiments, the vector can deliver the nucleic acid to target cells. In some embodiments, the nucleic acid-containing vector may be maintained in an episome. In some embodiments, the nucleic acid-containing vector may include a plasmid, minicircle DNA, virus, or any combination thereof. In some embodiments, the virus may include cytomegalovirus, adenovirus, or a combination thereof. In some embodiments, the nucleic acid-containing vector can be incorporated into the target cell genome via homologous recombination or random incorporation, e.g., via retrovirus-derived vectors such as MMLV, HIV-1, and ALV.

[0293] In some embodiments, the polynucleotide-derived polypeptide can be supplied to cells as a polypeptide. In some embodiments, the protein can be fused to a polypeptide domain that increases the solubility of the product. In some embodiments, the domain can be linked to the polypeptide via a defined protease cleavage site, such as a TEV sequence, which can be cleaved by a TEV protease. In some embodiments, the linker may include a flexible sequence. In some embodiments, the flexible sequence may include 1 to 10 glycine residues.

[0294] In some embodiments, the compositions disclosed herein may be operably linked to polypeptide permeability domains (e.g., covalently or non-covalently) to facilitate uptake by cells or organelles. In some embodiments, the polynucleotide compositions may include DNA, RNA, or a combination thereof. In some embodiments, this disclosure may relate to peptide-based polynucleotide carriers that may comprise two functional units: a polynucleotide-binding domain (e.g., a polycationic KH repeat domain) and a polypeptide permeability domain.

[0295] In some embodiments, several polypeptide permeable domains can be used in non-integrated polypeptides disclosed herein, including peptides, peptide mimes, non-peptide carriers, and any combination thereof. In some embodiments, the terms “permeable peptide,” “cell-permeable peptide,” “CPP,” “protein transduction domain,” and “PTD” can be used interchangeably herein. In some embodiments, the permeable peptide may be derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapaedia, called penetratin. In some embodiments, the CPP may include the amino acid sequences described in SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of International Patent Application PCT / US22 / 80942, which is incorporated herein by reference. In some embodiments, the permeable peptide may include, for example, the HIV-1 tat basic region amino acid sequence, which may include amino acids 49–57 of the naturally occurring tat protein. In some embodiments, the permeable domain may include a polyarginine motif. In some embodiments, the polyarginine motif may include the amino acid region 34–56 of the HIV-1 rev protein, nonaarginine, octaarginine, or any combination thereof. In some embodiments, a nonaarginine (R9) sequence may be used. In some embodiments, other cell-permeable peptides may include Pep-1, MPG, gamma-ZEIN, transportan, MAP, Pept1, Pept2, IVV-14, Ig(v), amphiphilic model peptides, pVEC, HRSV, Bp100 TAT2, or any combination thereof. In some embodiments, the compositions disclosed herein may be fused to a combination of polypeptide permeable domains. In some embodiments, the sites where fusion may occur may be selected to optimize the bioactivity, secretion, or binding properties of the polypeptide.

[0296] In some embodiments, the polynucleotide composition may comprise DNA, RNA, or any combination thereof. In some embodiments, the polynucleotide compositions disclosed herein may associate with a peptide-based polynucleotide carrier that may comprise an organelle-targeting signal. In some embodiments, for organelle-specific delivery, the peptide-based polynucleotide carrier may comprise two functional units: a polynucleotide-binding domain (e.g., a polycationic KH repeat domain) and an organelle-targeting peptide (e.g., a chloroplast transit peptide, a mitochondrial-targeting peptide).

[0297] This specification discloses compositions that can be prepared by in vitro synthesis. In some embodiments, various commercially available synthesis apparatuses can be used. In some embodiments, naturally occurring amino acids can be substituted with unnatural amino acids by using a synthesis apparatus. In some embodiments, specific sequences and preparation methods can be determined by convenience, cost-effectiveness, and required purity.

[0298] In some embodiments, if two or more different targeting complexes can be provided to cells (e.g., two different guide nucleic acids complementary to different sequences in the same or different target DNA), the complexes can be provided simultaneously (e.g., as two polypeptides and / or nucleic acids). In some embodiments, two or more different targeting complexes can be provided sequentially, for example, by first providing a targeting complex, then a second targeting complex, and vice versa.

[0299] In some embodiments, if the targeting complex and donor DNA can be supplied to the cells, they can be supplied simultaneously. In some embodiments, the targeting complex and donor DNA can be supplied sequentially, for example, by first supplying the targeting complex and then the donor DNA, and vice versa.

[0300] Methods of plant growth In some embodiments, a cell, plant, transgenic seed, progeny plant, or transgenic plant comprising one or more exogenous polynucleotides in the edited mitochondrial genome described herein can be grown in a temperature-controlled incubator, bioreactor, greenhouse, or a combination thereof. Optionally, the temperature-controlled incubator and / or greenhouse is further configured to control the light-dark cycle. In some embodiments, the cell, plant, transgenic seed, progeny plant, or transgenic plant can be grown in the dark for a predetermined period at a predetermined temperature. In some embodiments, the cell, plant, transgenic seed, progeny plant, or transgenic plant can be grown in the dark at 26 °C for 16-20 hours. In some embodiments, the plant, transgenic seed, progeny plant, or transgenic plant can be grown in a continuous light growth environment at 26-28 °C for root and shoot formation. In some embodiments, the plant, transgenic seed, progeny plant, or transgenic plant can be grown in a 16-hour / 8-hour light / dark growth chamber at 26-28 °C for root and shoot formation. In some embodiments, a progeny plant or transgenic plant showing both root and shoot development can be transferred to a pot containing an artificial potting medium and gradually acclimated to greenhouse conditions. In some embodiments, the plant, transgenic seed, progeny plant, or transgenic plant can be grown in a field. In some embodiments, the field can be treated with an inhibitor of acetolactate synthase.

[0301] Compositions and Kits This specification also provides compositions comprising any of the polynucleotides, polypeptides, vectors, or reagents (e.g., phosphites) described herein. Each composition may comprise any of the polynucleotides, polypeptides, vectors, or reagents described herein, and one or more (e.g., 1, 2, 3, 4, or 5) acceptable carriers or diluents. In some embodiments, the kit may comprise cells, tissues, reproductive materials, seeds, pollen, offspring, or any combination thereof.

[0302] In some embodiments, any of the compositions described herein may include one or more buffers (e.g., neutral buffered saline, phosphate-buffered saline (PBS)), one or more growth regulators (e.g., naphthaleneacetic acid, 6-benzylaminopurine, phytagel), and one or more culture media (e.g., germination medium, growth medium, maturation medium, phosphyte medium).

[0303] In some embodiments, any of the compositions described herein may further include one or more agents (e.g., 1, 2, 3, 4, or 5) that facilitate the entry of any of the vectors or nucleic acids described herein into cells (e.g., plant cells).

[0304] In some embodiments, either the vectors or nucleic acids described herein can be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that may be included in any of the pharmaceutical compositions described herein include, but are not limited to, poloxamers, chitosans, dendrimers, and poly(lactic acid-co-glycolic acid) (PLGA) polymers.

[0305] Kits are also provided that include any of the compositions described herein, which include any of the polynucleotides, polypeptides, reagents, or vectors described herein.

[0306] In some embodiments, the kit may include instructions for performing any of the methods described herein.

[0307] Exemplary Embodiments The following non-limiting embodiments provide examples of the present invention, but do not limit the scope of the invention.

[0308] Embodiment 1. A method for transforming mitochondria, a) A step of producing transformed mitochondria by introducing a first polynucleotide encoding a first polypeptide into the mitochondria of a cell that is a plant cell or an algal cell, wherein the first polypeptide has herbicide-resistant acetolactate synthase activity. b) A step of growing cells under conditions in which the first polypeptide is expressed, c) A step of growing cells in a culture medium containing an acetolactate synthase inhibitor, d) A method comprising the step of selecting cells containing transformed mitochondria containing a first polynucleotide.

[0309] Embodiment 2. A method for transforming mitochondria, a) In the mitochondria of a plant cell or algal cell, i) A first polynucleotide encoding a first polypeptide for generating transformed mitochondria, wherein the first polypeptide has herbicide-resistant acetolactate synthase activity, and ii) A step of introducing additional polynucleotides that encode selectable markers, b) A step of growing cells under conditions in which a selectable marker is expressed, c) A step of growing cells in a culture medium containing a selectable marker, d) A method comprising the step of selecting cells containing transformed mitochondria containing a first polynucleotide.

[0310] Embodiment 3. The method according to Embodiment 2, wherein the selectable marker codes for a product that provides resistance to other toxic compounds.

[0311] Embodiment 4. The method according to Embodiment 2, wherein the selectable marker is a phosphyte dehydrogenase enzyme or a biologically active fragment thereof, and the selector is a phosphyte.

[0312] Embodiment 5. A method for transforming mitochondria, comprising the step of introducing a first heterologous polynucleotide encoding a first polypeptide into the mitochondria of a cell, wherein the cell includes a plant cell or an algal cell, and the first polypeptide includes an acetolactate synthase enzyme or a biologically active fragment thereof.

[0313] Embodiment 6. The method according to Embodiment 5, wherein the acetolactate synthase enzyme or a biologically active fragment thereof has herbicide resistance activity.

[0314] Embodiment 7. The method according to Embodiment 5 or 6, further comprising the step of growing cells under conditions in which the first polypeptide is expressed.

[0315] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the transformed mitochondria comprises an edited mitochondrial genome containing a first polynucleotide.

[0316] Embodiment 9. The method according to any one of Embodiments 5 to 8, further comprising the step of growing cells in a culture medium containing an acetolactate synthase inhibitor.

[0317] Embodiment 10. The method according to Embodiment 2, wherein the selective agent comprises an acetolactate synthase inhibitor.

[0318] Embodiment 11. The method according to Embodiments 1, 9, or 10, wherein the acetolactic acid synthase inhibitor comprises sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl benzoate, sulfonanilide, sulfonylaminocarbonyltriazolinone, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

[0319] Embodiment 12. The method according to Embodiment 11, wherein the sulfonylurea contains chlorsulfuron.

[0320] Embodiment 13. The method according to any one of Embodiments 1 to 4 or 9 to 12, wherein the culture medium contains chlorsulfuron at a concentration of 20 nM to 100 nM, 100 nM to 1 μM, 1 μM to 20 μM, or 20 μM to 100 μM.

[0321] Embodiment 14. The method according to Embodiment 11, wherein the imidazolinone comprises imazapyr, imazapic, imazetapyr, imazamox, imazametabenz, imazakine, any salt thereof, any stereoisomer thereof, or any combination thereof.

[0322] Embodiment 15. The method according to Embodiment 11, wherein the triazolopyrimidine comprises penoxyslam, chloranslam-methyl, dicloslam, floraslam, flumethoslam, metoslam, pyroxyslam, any salt thereof, any stereoisomer thereof, or any combination thereof.

[0323] Embodiment 16. The method according to Embodiment 11, wherein the pyrimidinyl benzoate comprises bispyribac sodium, pyribenzoxime, pyrithiobac sodium, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

[0324] Embodiment 17. The method according to Embodiment 11, wherein the sulfonanilide comprises pyrimisulfan, triafamone, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

[0325] Embodiment 18. The method according to Embodiment 11, wherein the sulfonylaminocarbonyltriazolinone comprises fulcarbazone-sodium, propoxycarbazone-sodium, thiencarbazone-methyl, any salt thereof, any stereoisomer thereof, or any combination thereof.

[0326] Embodiment 19. Sulfonylurea is amidesulfuron, azimsulfuron, bensulfuron-methyl, chlorimulon-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, etamethosulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron-methyl-na, horamsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron-methyl-na, mesosulfuron-methyl, metazosulfuron, metosulfuron-methyl, nicosulfuron The method according to Embodiment 11, which is lon, orthosulfamulone, oxasulfurone, primisulfurone-methyl, propyrisulfurone, prosulfurone, pyrazosulfurone-ethyl, limsulfurone, sulfomethurone-methyl, sulfosulfurone, thifensulfurone-methyl, triasulfurone, tribenulon-methyl, trifloxysulfurone-na, triflusulfurone-methyl, tritosulfurone, any salt of any of these, any stereoisomer of any of these, or any combination thereof.

[0327] Embodiment 20. The method according to any one of Embodiments 1 to 19, further comprising the steps of introducing a second polynucleotide encoding a regulatory subunit of acetolactate synthase or a biologically active fragment thereof into mitochondria, and growing cells under conditions in which the second polypeptide is expressed.

[0328] Embodiment 21. The method according to any one of Embodiments 1 to 19, further comprising the steps of introducing a third polynucleotide encoding a modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof into the nucleus of a cell, wherein the modified regulatory subunit of acetolactate synthase or a modified biologically active fragment comprises a mitochondrial-targeting peptide, and growing the cell under conditions in which the third polypeptide is expressed.

[0329] Embodiment 22. The method according to Embodiment 21, wherein the third polynucleotide encodes a third polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 21.

[0330] Embodiment 23. The method according to Embodiment 20, further comprising the step of selecting cells, wherein the transformed mitochondria contain a second polynucleotide.

[0331] Embodiment 24. The method according to any one of Embodiments 21 to 22, further comprising the step of selecting cells containing transformed nuclei, wherein the transformed nuclei contain a third polynucleotide.

[0332] Embodiment 25. The method according to any one of Embodiments 1 to 24, wherein the cells are plant cells selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, and soybean cells.

[0333] Embodiment 26. A step of introducing donor DNA into the mitochondria of a cell, wherein the donor DNA is a) A fourth polynucleotide that is heterogeneous to mitochondria, b) The fifth polynucleotide at the first end, c) The sixth polynucleotide at the second end The process includes a step in which the fifth polynucleotide and the sixth polynucleotide each contain sequences capable of homologous recombination with the endogenous mitochondrial DNA sequence, and homologous recombination between all or part of the donor DNA and the endogenous mitochondrial DNA sequence incorporates the fourth polynucleotide into the endogenous mitochondrial DNA sequence. The method according to any one of Embodiments 1 to 25, further comprising the step of selecting cells having an edited mitochondrial genome, wherein the edited mitochondrial genome comprises a fourth polynucleotide.

[0334] Embodiment 27. The method according to Embodiment 26, wherein the donor DNA comprises a first polynucleotide, or both a first polynucleotide and a second polynucleotide.

[0335] Embodiment 28. The method according to Embodiment 27, wherein the edited mitochondrial genome comprises a first polynucleotide.

[0336] Embodiment 29. The method according to Embodiment 28, wherein the edited mitochondrial genome contains a second polynucleotide.

[0337] Embodiment 30. The method according to Embodiment 26, wherein the donor DNA does not contain the first polynucleotide.

[0338] Embodiment 31. The method according to Embodiment 30, wherein the donor DNA does not contain a second polynucleotide.

[0339] Embodiment 32. The method according to any one of Embodiments 26 to 31, wherein the fourth polynucleotide encodes a fourth polypeptide or a functional RNA, or both.

[0340] Embodiment 33. The method according to any one of Embodiments 26 to 32, wherein the fourth polynucleotide comprises a cytoplasmic male sterility (CMS) coding region.

[0341] Embodiment 34. The method according to Embodiment 33, wherein the CMS code area includes orf79.

[0342] Embodiment 35. The method according to Embodiment 33, wherein the CMS code region encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to sequence number 47.

[0343] Embodiment 36. The method according to Embodiment 34 or 35, wherein the cells are rice cells.

[0344] Embodiment 37. The method according to Embodiment 33, wherein the CMS code area includes orf256 or orf279.

[0345] Embodiment 38. The method according to Embodiment 33, wherein the CMS code region encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to sequence number 54.

[0346] Embodiment 39. The method according to Embodiment 33, wherein the CMS code region encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to sequence number 56.

[0347] Embodiment 40. The method according to any one of Embodiments 37 to 39, wherein the cells are wheat cells.

[0348] Embodiment 41. The method according to any one of Embodiments 26 to 40, wherein the homologous recombination-capable sequence in the fifth polynucleotide has a size of 25-75 nucleotides, 25-100 nucleotides, 25-150 nucleotides, 25-200 nucleotides, 25-300 nucleotides, 25-400 nucleotides, 25-500 nucleotides, 25-1000 nucleotides, 25-1500 nucleotides, or 25-2000 nucleotides.

[0349] Embodiment 42. The method according to any one of Embodiments 26 to 41, wherein the homologous recombination-capable sequence in the sixth polynucleotide has a size of 25-75 nucleotides, 25-100 nucleotides, 25-150 nucleotides, 25-200 nucleotides, 25-300 nucleotides, 25-400 nucleotides, 25-500 nucleotides, 25-1000 nucleotides, 25-1500 nucleotides, or 25-2000 nucleotides.

[0350] Embodiment 43. The method according to any one of Embodiments 26 to 42, wherein at least one selected from the group consisting of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, a sixth polynucleotide, and any combination thereof is introduced into cells via microinjection, meristematic transformation, electroporation, Agrobacterium-mediated transformation, virus-based gene transfer, transfection, vacuum infiltration, particulate gun bombardment, or any combination thereof.

[0351] Embodiment 44. The method according to any one of Embodiments 26 to 43, wherein at least one selected from the group consisting of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, a sixth polynucleotide, and any combination thereof is introduced into a cell as a peptide-polynucleotide complex, the peptide-polynucleotide complex comprising at least one peptide.

[0352] Embodiment 45. The method according to Embodiment 44, wherein at least one peptide of the peptide-polynucleotide complex comprises at least one selected from the group consisting of cell-permeable peptides (CPPs), organelle-targeting peptides, mitochondrial-targeting peptides, histidine-rich peptides, lysine-rich peptides, and any combination thereof.

[0353] Embodiment 46.a. In the mitochondria of a cell, i. A first additional polynucleotide encoding at least one guide polynucleotide, wherein at least one guide polynucleotide guides the polynucleotide-derived polypeptide to cleave at least one target sequence present in the organelle genome, A second additional polynucleotide encoding a polynucleotide-derived polypeptide, wherein the polynucleotide-derived polypeptide, when associated with the guide polynucleotide, cleaves at least one target sequence, and The method according to any one of embodiments 1 to 45, further comprising the step of introducing a recombinant DNA construct containing the above.

[0354] Embodiment 47.a. In the nucleus of a cell, i) A first additional polynucleotide encoding a modified polynucleotide-derived polypeptide, wherein the modified polynucleotide-derived polypeptide comprises a polynucleotide-derived polypeptide operably linked to a mitochondrial targeting peptide, and the first additional polynucleotide cleaves at least one target sequence present in the mitochondrial genome when the polynucleotide-derived polypeptide is associated with a guide RNA. ii) A second additional polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA guides the polynucleotide-derived polypeptide to cleave at least one target sequence present in the mitochondrial genome. The method according to any one of embodiments 1 to 45, further comprising the step of introducing [a certain element].

[0355] Embodiment 48.a. In the nucleus of a cell, i. A step of introducing a first additional polynucleotide encoding a modified polynucleotide-derived polypeptide, wherein the modified polynucleotide-derived polypeptide comprises a polynucleotide-derived polypeptide operably linked to a mitochondrial targeting peptide, and the polynucleotide-derived polypeptide, when associated with a guide RNA, cleaves at least one target sequence present in the mitochondrial genome. b. In the mitochondria of cells, i. A second additional polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA guides the polynucleotide-derived polypeptide to cleave at least one target sequence present in the mitochondrial genome. The method according to any one of embodiments 1 to 45, further comprising the step of introducing a

[0356] Embodiment 49. The method according to any one of Embodiments 46 to 48, wherein the polynucleotide-derived polypeptide is at least one selected from the group consisting of Cas9 protein, Cas3 protein, MAD2 protein, MAD7 protein, CRISPR nuclease, nuclease domain of Cas protein, Cpf1 protein, Argonaut, modified forms thereof, biologically active fragments thereof, and any combination thereof.

[0357] Embodiment 50. The method according to any one of Embodiments 46-49, wherein homologous recombination of all or part of donor DNA with an endogenous mitochondrial DNA sequence results in an edited mitochondrial genome lacking at least one target sequence.

[0358] Embodiment 51. The method according to any one of Embodiments 46 to 50, further comprising the step of introducing a third additional polynucleotide into the nucleus of a cell, wherein the third additional polynucleotide encodes a modified site-specific nuclease, the modified site-specific nuclease comprises a site-specific nuclease operably linked to a mitochondrial-targeting peptide, and the site-specific nuclease cleaves at least one target sequence present in the mitochondrial genome.

[0359] Embodiment 52. The method according to Embodiment 51, wherein the site-specific nuclease is at least one selected from the group consisting of TALENs, zinc finger nucleases, meganucleases, restriction enzymes, and any combination thereof.

[0360] Embodiment 53. The method according to any one of Embodiments 1 to 52, wherein the first polynucleotide encoding the first polypeptide further comprises a T7 RNA polymerase promoter, and the expression of the first polypeptide is under the control of the T7 RNA polymerase promoter.

[0361] Embodiment 54. The method according to any one of Embodiments 1 to 53, further comprising the step of introducing a fourth additional polynucleotide encoding a modified T7 RNA polymerase into the nucleus of a cell, wherein the modified T7 RNA polymerase comprises a T7 RNA polymerase operably linked to a mitochondrial-targeting peptide.

[0362] Embodiment 55. The method according to any one of Embodiments 1 to 54, wherein the first polypeptide comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 24.

[0363] Embodiment 56. The method according to any one of Embodiments 1 to 55, wherein the first polypeptide comprises SEQ ID NO: 24.

[0364] Embodiment 57. The method according to any one of Embodiments 1 to 56, wherein the first polynucleotide encoding the first polypeptide has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 25.

[0365] Embodiment 58. The method according to any one of Embodiments 1 to 57, wherein the first polynucleotide comprises SEQ ID NO: 25.

[0366] Embodiment 59. The method according to any one of Embodiments 1 to 58, wherein the sequence encoding the start codon of the first polypeptide is replaced with the sequence encoding the mitochondrial RNA editing site.

[0367] Embodiment 60. The method according to Embodiment 59, wherein the mitochondrial RNA editing site is derived from the mitochondrial nad4L gene or the mitochondrial cox2 gene.

[0368] Embodiment 61. The method according to Embodiment 60, wherein the sequence encoding the mitochondrial RNA editing site includes SEQ ID NO: 41 or SEQ ID NO: 42.

[0369] Embodiment 62. The method according to any one of Embodiments 1 to 61, further comprising the steps of introducing a fifth additional polynucleotide encoding an additional selectable marker polypeptide into the nucleus of a cell, wherein the additional selectable marker polypeptide confers resistance to an additional selector, and selecting cells that grow in the presence of the additional selector.

[0370] Embodiment 63. The method according to Embodiment 62, wherein cells are grown simultaneously in the presence of an additional selective agent and in the presence of an acetolactate synthase inhibitor.

[0371] Embodiment 64. The method according to Embodiment 62, wherein cells are grown sequentially, first in the presence of an additional selective agent, and then in the presence of an acetolactate synthase inhibitor.

[0372] Embodiment 65. The method according to any one of Embodiments 62 to 64, wherein the additional selectable marker polypeptide is a polypeptide having hygromycin phosphotransferase (HPT) activity, and the additional selector is hygromycin.

[0373] Embodiment 66. The method according to any one of Embodiments 26 to 65, further comprising the step of removing the first polynucleotide encoding the first polypeptide from the transformed mitochondria after the incorporation of the fourth polynucleotide.

[0374] Embodiment 67. The method according to any one of Embodiments 8 to 66, comprising the step of selecting cells containing multiple mitochondrial genomes, wherein at least 50%, 60%, 70%, 80%, 90%, or 100% of the multiple mitochondrial genomes contain edited mitochondrial genomes.

[0375] Embodiment 68. The method according to any one of Embodiments 8 to 67, further comprising the step of selecting cells that are isocytoplasmic to the edited mitochondrial genome.

[0376] Embodiment 69. The method according to any one of Embodiments 1 to 68, wherein the cells are plant cells, and a plant grows from the plant cells.

[0377] Embodiment 70. The method according to Embodiment 69, further comprising the step of selecting a plant containing the first polypeptide.

[0378] Embodiment 71. A cell produced by the method described in any one of Embodiments 1 to 70, which is a plant cell selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, and soybean cells.

[0379] Embodiment 72. Plants, cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, produced from plant cells as described in Embodiment 71, wherein the cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, include an edited mitochondrial genome.

[0380] Embodiment 73. A method for controlling weeds, comprising the step of growing a plurality of plants in the presence of an acetolactate synthase inhibitor, wherein at least one of the plurality of plants contains mitochondria containing heterologous polynucleotides encoding a polypeptide having herbicide-resistant acetolactate synthase activity, and the presence of the acetolactate synthase inhibitor selectively promotes the growth of at least one of the plurality of plants, and is sufficient to increase the growth of at least one of the plurality of plants compared to plants lacking polynucleotides encoding a polypeptide having herbicide-resistant acetolactate synthase activity.

[0381] Embodiment 74. The method according to Embodiment 73, further comprising the step of applying an acetolactate synthase inhibitor to a plant, multiple plants, soil adjacent to a plant, or any combination thereof.

[0382] Embodiment 75. The method according to Embodiment 74, wherein an acetolactate synthase inhibitor is applied as a foliar fertilizer.

[0383] Embodiment 76. The method according to Embodiment 74, wherein an acetolactate synthase inhibitor is applied as a soil conditioner.

[0384] Embodiment 77. The method according to any one of Embodiments 73 to 76, wherein at least one of the multiple plants is selected from the group consisting of wheat, corn, rice, barley, sorghum, rye, sugarcane, potato, tomato, canola, broccoli, cauliflower, and soybean.

[0385] Embodiment 78. The method according to any one of Embodiments 73 to 77, wherein the plant lacking a polynucleotide encoding a polypeptide having herbicide-resistant acetolactate synthase activity is a weed.

[0386] Embodiment 79. The method according to any one of Embodiments 73 to 78, wherein the polypeptide having herbicide-resistant acetolactate synthase activity comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with SEQ ID NO: 24.

[0387] Embodiment 80. The method according to Embodiment 78, wherein the polypeptide having acetolactate synthase activity comprises the amino acid sequence of SEQ ID NO: 24.

[0388] Embodiment 81. A cell comprising an edited mitochondrial genome, wherein the cell is a plant cell or an algal cell, and the edited mitochondrial genome comprises heterologous polynucleotides encoding polypeptides having herbicide-resistant acetolactate synthase activity.

[0389] Embodiment 82. The cell according to Embodiment 91, which is a plant cell selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, and soybean cells.

[0390] Embodiment 83. The cell according to Embodiment 81 or Embodiment 82, wherein the edited mitochondrial genome comprises at least one nucleotide substitution, deletion, or insertion.

[0391] Embodiment 84. A cell according to any one of Embodiments 81 to 83, comprising transformed mitochondria containing an edited mitochondrial genome.

[0392] Embodiment 85. A cell according to any one of Embodiments 81 to 84, wherein the amino acid sequence of a polypeptide having herbicide-resistant acetolactate synthase activity encoded by heterologous polynucleotides contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 24.

[0393] Embodiment 86. The cell according to Embodiment 85, wherein the amino acid sequence of the polypeptide having herbicide-resistant acetolactate synthase activity comprises SEQ ID NO: 24.

[0394] Embodiment 87. The cell according to Embodiment 86, wherein a heterologous polynucleotide encoding a polypeptide having herbicide-resistant acetolactate synthase activity comprises SEQ ID NO: 25.

[0395] Embodiment 88. A cell according to any one of Embodiments 81 to 87, wherein the sequence encoding the start codon of a heterogeneous polynucleotide is replaced with the sequence encoding a mitochondrial RNA editing site.

[0396] Embodiment 89. The cell according to Embodiment 88, wherein the mitochondrial RNA editing site is derived from the mitochondrial nad4L gene or the mitochondrial cox2 gene.

[0397] Embodiment 90. The cell according to Embodiment 89, wherein the sequence encoding the mitochondrial RNA editing site includes SEQ ID NO: 41 or SEQ ID NO: 42.

[0398] Embodiment 91. A cell according to any one of Embodiments 81 to 90, wherein the edited mitochondrial genome further comprises a second polynucleotide encoding a polypeptide or a functional RNA, or both, and the polypeptide and functional RNA are heterogeneous to the mitochondria.

[0399] Embodiment 92. The cell according to Embodiment 91, wherein the second polynucleotide includes a cytoplasmic male sterility (CMS) coding region.

[0400] Embodiment 93. The cell according to Embodiment 92, wherein the CMS code region is orf79.

[0401] Embodiment 94. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 47.

[0402] Embodiment 95. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide containing SEQ ID NO: 47.

[0403] Embodiment 96. A cell according to any one of Embodiments 92 to 95, which is a rice cell.

[0404] Embodiment 97. The cell according to Embodiment 92, wherein the CMS code region is orf256 or orf279.

[0405] Embodiment 98. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 54.

[0406] Embodiment 99. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide containing SEQ ID NO: 54.

[0407] Embodiment 100. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% sequence identity with respect to SEQ ID NO: 56.

[0408] Embodiment 101. The cell according to Embodiment 91, wherein the second polynucleotide encodes a polypeptide comprising SEQ ID NO: 56.

[0409] Embodiment 102. A cell according to any one of Embodiments 97 to 101, which is a wheat cell.

[0410] Embodiment 103. A cell according to any one of Embodiments 81 to 102, further comprising a third heterologous polynucleotide in the nucleus of the cell, wherein the third heterologous polynucleotide encodes an additional selectable marker polypeptide that gives the cell resistance to an additional selective agent.

[0411] Embodiment 104. The cells according to Embodiment 103, wherein an additional selectable marker polypeptide has hygromycin phosphotransferase (HPT) activity.

[0412] Embodiment 105. The cells according to Embodiment 104, wherein the additional selective agent is hygromycin.

[0413] Embodiment 106. A cell according to any one of Embodiments 81 to 105, comprising multiple mitochondrial genomes, wherein at least 50%, 60%, 70%, 80%, 90%, or 100% of the multiple mitochondrial genomes comprises edited mitochondrial genomes.

[0414] Embodiment 107. A cell according to any one of Embodiments 81 to 106, which is isocytoplasmic to an edited mitochondrial genome.

[0415] Embodiment 108. A cell according to any one of Embodiments 81 to 107, expressing a polypeptide having herbicide-resistant acetolactate synthase activity.

[0416] Embodiment 109. A cell according to any one of Embodiments 81 to 96, wherein the edited mitochondrial genome comprises a fourth heterologous polynucleotide encoding a regulatory subunit of acetolactate synthase or a biologically active fragment thereof.

[0417] Embodiment 110. The cell according to Embodiment 109, expressing a regulatory subunit of acetolactic acid synthase encoded by a fourth heterologous polynucleotide or a biologically active fragment thereof.

[0418] Embodiment 111. A cell according to any one of Embodiments 81 to 108, wherein the cell nucleus comprises a fifth heterologous polynucleotide encoding a modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof, and the modified regulatory subunit of acetolactate synthase or a modified biologically active fragment comprises a mitochondrial-targeting peptide.

[0419] Embodiment 112. The cell according to Embodiment 111, expressing a modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof.

[0420] Embodiment 113. Cells according to any one of Embodiments 81 to 112, grown in a culture medium containing an acetolactate synthase inhibitor.

[0421] Embodiment 114. The cell according to Embodiment 113, wherein the polypeptide having herbicide-resistant acetolactate synthase activity exhibits resistance to at least one herbicide selected from the group consisting of sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl (thio)benzoate, sulfonanilide, sulfonylaminocarbonyltriazolinone, and any combination thereof.

[0422] Embodiment 115. The cell according to Embodiment 114, wherein the polypeptide having herbicide-resistant acetolactate synthase activity exhibits resistance to sulfonylurea.

[0423] Embodiment 116. The cell according to Embodiment 115, wherein the sulfonylurea is chlorsulfuron.

[0424] Embodiment 117. The cell according to Embodiment 116, wherein the polypeptide having herbicide-resistant acetolactate synthase activity exhibits resistance to chlorsulfuron at concentrations of at least 20 nM to 100 nM, 100 nM to 1 μM, 1 μM to 20 μM, or 20 μM to 100 μM.

[0425] Embodiment 118. A transgenic plant or a portion thereof comprising the cells described in any one of Embodiments 81 to 117.

[0426] Embodiment 119. The transgenic plant or part thereof according to Embodiment 118, further comprising cells, tissues, reproductive material, seeds, pollen, offspring, or any combination thereof.

[0427] Embodiment 120. A transgenic plant or part thereof according to Embodiment 118 or 119, grown in a temperature-controlled incubator.

[0428] Embodiment 121. The transgenic plant or part thereof according to Embodiment 120, further comprising a temperature-controlled incubator with a light-dark cycle.

[0429] Embodiment 122. A field or greenhouse containing the transgenic plant or a part thereof as described in Embodiment 118.

[0430] Embodiment 123. A food comprising the cells described in any one of Embodiments 81 to 117.

[0431] Embodiment 124. A field containing cells according to any one of Embodiments 81 to 117.

[0432] Embodiment 125. A kit comprising cells according to any one of Embodiments 81 to 117 or a transgenic plant or part thereof according to any one of Embodiments 118 to 121. [Examples]

[0433] This disclosure is further defined by the following embodiments, and unless otherwise specified, parts and percentages are in units of weight and degrees are in degrees Celsius. These embodiments illustrate embodiments, but should be understood as being given only as examples. From the above considerations and these embodiments, the essential features of this disclosure can be identified, and this disclosure can be adapted to various uses and conditions by considering various changes and modifications to this disclosure without departing from its spirit and scope. Such modifications are also intended to fall within the scope of the appended claims.

[0434] Example 1 Chlorsulfuron selection in transformed rice cells Embryogenic callus cultures of wild-type rice varieties were initiated and maintained for at least 4–6 weeks in Chu-N6-based callus induction and maintenance medium supplemented with plant growth regulator 2,4-D. Prior to transformation, the callus cultures were subcultured for 4 days in fresh N6-based callus maintenance medium. Approximately 4 hours before transformation, callus was prepared for transformation by seeding tissue into the target area on the same N6-based medium supplemented with mannitol and sorbitol for osmotic protection.

[0435] Rice callus was transformed with various ALS expression constructs (e.g., herbicide-resistant ALS large subunit, regulatory ALS small subunit) using particle bombardment. The following steps were used for cultivation, selection, and regeneration.

[0436] 1. After bombardment, the callus was incubated at 26°C for 16–20 hours in the dark, and then the callus tissue aggregates, approximately 1–3 mm in size, were subcultured in selective medium, which was a callus maintenance medium supplemented with 20–100 nM chlorsulfuron and 25–50 mg / L hygromycin appropriate for the bombardmented genes. The callus on the selective medium was then returned to incubation in the dark for 2–3 weeks.

[0437] 2. After incubation in the dark for 2-3 weeks, small (1-3 mm) aggregates were subcultured again in fresh selective medium containing chlorsulfuron and incubated for approximately 2 weeks in a plant growth chamber illuminated at 26°C with a light cycle of 16 hours light to 8 hours dark, a light intensity of 60 μmol / m² / second. After a 2-week maintenance period under light, further subculture in fresh selective medium was the most common method.

[0438] 3. At the end of the second selection period or thereafter, 5–8 weeks after bombardment, actively growing calluses (individual initial events) were separated from the surrounding dead tissue and transferred to individual plates of fresh selective medium supplemented with chlorsulfuron to maintain their individual identity. Where many initial events were present, each event occupied one-eighth to half of the plate, and the identity of each event was maintained separately. In some cases, moderately or poorly growing calluses were also maintained for comparison.

[0439] Multiple selection processes When the ALS gene expression cassette was transformed simultaneously with the 35S:HPT nuclear expression cassette conferring hygromycin B resistance, event selection was facilitated by using 25-50 mg / L of hygromycin B along with chlorsulfuron in the selection medium. For optimal recovery of the ALS gene-expressing event, the timing of introducing hygromycin selection in combination with chlorsulfuron selection was modified.

[0440] In some experiments (with or without 35S:HPT co-expression), herbicide-resistant ALS gene expression cassettes were also linked and transformed simultaneously with oliR expression cassettes that confer resistance to the antibiotic oligomycin. In some experiments, the geminivirus VOR sequence (target site of the geminivirus Rep protein) was also present in the construct. In some experiments, oligomycin was incorporated into the selective medium at a rate of 1–5 mg / L. In some experiments where oligomycin selection was included, the carbon source sucrose was reduced to 0.5% (5 mg / L) or replaced with 60 ml of sterile 50% glycerol solution per liter of medium to improve the efficacy of oligomycin. In some experiments where oligomycin was the selective agent, the compound disulfiram was also incorporated into the medium at 100 μM to inhibit the cells' ability to utilize all the alcohol produced by anaerobic respiration of the treated cells.

[0441] In some experiments, the timing of initiating oligomycin selection in combination with chlorsulfuron selection was modified. In other experiments, after the initial period of one or more passage and selection cycles, the use of chlorsulfuron was discontinued, and oligomycin alone was used at the above concentrations.

[0442] Sampling for molecular analysis Approximately 10 weeks or more after selection, samples from individual events were collected for PCR analysis. One or more small tissue aggregates, approximately 1 mm in diameter, were supplied for each initial event to be analyzed. Maintenance and sampling of callus events were continued for 6–8 months.

[0443] Embryonic maturation and plant regeneration 4. In some experiments, at the end of proliferation for more than two weeks in individual initial events, callus that maintained growth (representing a presumed mitochondrial transformation event) was transferred to an N6-based medium for embryo maturation that still contained chlorsulfuron as a selective agent, but from which growth regulator 2,4-D had been removed and supplemented with 2.5 g / L Phytagel.

[0444] 5. After at least 10–14 days, mature somatic embryos showing signs of normal maturation were transferred to an N6-based germination medium still containing chlorsulfuron as a selective agent. This medium was supplemented with the growth regulators 0.2 mg / L naphthaleneacetic acid and 2 mg / L 6-benzylaminopurine, as well as 2.5 g / L Phytagel. These embryos were grown at 26°C in a 16-hour / 8-hour light / dark growth chamber to form roots and shoots.

[0445] 6. After step 5, plants showing development of both roots and shoots were transferred to pots containing artificial potting medium for acclimatization and grown in a greenhouse.

[0446] Inducible expression system In some experiments, the target tissue for rice microparticle gun transformation was derived from different sources. Different sources of callus tissue were derived from the previously described Agrobacterium tumefaciens transformation. This transformation induced an event with a dexamethasone-inducible system for producing the Geminivirus Rep protein. Inducible transgenic lines were identified by pre-screening using RFP visual markers as described in Example 6. To supply sufficient tissue for bombardment, this event was maintained on a first selective medium supplemented with 40 mg / L hygromycin, amino acids, proline, maltose, and 2,4-D growth regulator for approximately two months prior to microparticle gun transformation.

[0447] Tissues targeted for bombardment were pre-cultured for 4 days on the first select medium before bombardment when derived from the inducing line. In some experiments, this pre-culture medium was further supplemented with 1,000 μl of 10 μM dexamethasone (DEX) dissolved in DMSO, or 1,000 μl of DMSO alone as a negative control. DEX was included as a chemical inducer of the Rep protein.

[0448] Approximately four hours before transformation, callus was prepared for bombardment by seeding tissue into the target region on an N6-based medium that contained neither DEX nor DMSO, but was supplemented with mannitol and sorbitol for osmotic protection.

[0449] 1. After bombardment, the callus was incubated in the dark at 26°C for 16–20 hours. Then, as described above, and appropriate for the bombarded genes, aggregates of callus tissue approximately 1–3 mm in size were subcultured in N6-based callus maintenance medium supplemented with growth regulator 2,4-D and appropriate selectors containing 20–100 nM chlorsulfuron, 25–50 mg / L hygromycin, and 1–5 mg / L oligomycin. In some experiments, chemical induction with DEX in the medium was initiated during the initial selection. The callus on the selective medium with DEX and / or DMSO was then returned to incubation in the dark for the initial selection. In some experiments, DEX and / or DMSO were introduced at a later point in the selection process.

[0450] 2. After incubation in the dark for 2-3 weeks, small (1-3 mm) aggregates were subcultured again in fresh selective medium and then incubated under light as described above. After maintaining under light for 2-3 weeks, further subculture in fresh selective medium containing DEX and / or DMSO was most frequently performed.

[0451] 3. In some experiments, induction with DEX was continued throughout the entire selection process. In some experiments, the use of DEX and / or DMSO was discontinued for one or more culture periods. In some experiments, DEX was reintroduced at a point later in the selection process.

[0452] Sampling for molecular analysis Approximately 10 weeks or more after selection, samples from individual events were collected for PCR analysis. One or more small tissue aggregates, approximately 1 mm in diameter, were supplied for each initial event to be analyzed. Maintenance and sampling of callus events were continued for 6–8 months.

[0453] Example 2 Sulfonylurea selection of transformed cells by transfer of herbicide-resistant ALS large subunit protein into rice mitochondria The use of herbicide-resistant acetolactate synthase (ALS) as a selectable marker for mitochondrial transformation was tested by transferring the protein, produced in the cytoplasm, into the mitochondria of rice callus cells. In this experiment, the inventors constructed plasmid pNAP170 containing the following three polypeptides: 1) MTS-ALS(HR-LS) (herbicide-resistant ALS catalytic large subunit ALS(HR-LS) fused with the mitochondrial targeting sequence (MTS) of the Arabidopsis rsp10 protein), 2) MTS-ALS(SS) (regulatory small subunit ALS(SS) fused with the mitochondrial targeting sequence (MTS) of the Arabidopsis At5g47030 gene), and 3) HPT (hygromycin phosphotransferase protein for use as a selectable marker for nuclear transformation).

[0454] To produce the MTS-ALS(HR-LS) protein, we used a polynucleotide sequence (SEQ ID NO: 7, rice gene ID: AB049823) encoding the rice herbicide-resistant ALS large subunit polypeptide, which has been shown to confer sulfonylurea (SU) resistance to rice (Plant Biotech. 27:75 by Kawai et al.). The encoded protein contains a chloroplast-targeting sequence. To target this to mitochondria, we deleted the sequence encoding the first 18aa residues (SEQ ID NO: 8), which consists of the chloroplast-targeting sequence, and replaced it with the sequence encoding the mitochondrial-targeting sequence of the Arabidopsis mitochondrial ATP synthase subunit delta protein (SEQ ID NO: 9, gene ID: At5g47030). The corresponding DNA fragments with adjacent BamHI and KpnI restriction sites were synthesized by the external supplier GENEWIZ®. The nucleotide sequence encoding the MTS-ALS(HR-LS) protein is presented as SEQ ID NO: 10.

[0455] The amino acid sequence of the MTS-ALS(HR-LS) protein is presented as Sequence ID No. 11. The first 56 amino acids of Sequence ID No. 11 correspond to the MTS of the Arabidopsis saliana rsp10 protein.

[0456] Synthetic DNA containing the coding region for MTS-ALS(HR-LS) was cloned into pNAP148, and an expression cassette was constructed having the MTS-ALS(HR-LS) coding region operably ligated to the maize UBI1 promoter and intron (SEQ ID NO: 12) and NOS terminator (SEQ ID NO: 13). The resulting plasmid pNAP152 further contains an expression cassette having a nucleotide sequence encoding hygromycin phosphotransferase (SEQ ID NO: 14) operably ligated to the 35S promoter (SEQ ID NO: 15) and CaMV terminator (SEQ ID NO: 16).

[0457] The activity of the ALS catalytic large subunit protein has been shown to be enhanced by the presence of a small subunit protein, which the inventors have named ALS(SS). In experiments with rice, the inventors identified a rice homolog of ALS(SS) (SEQ ID NO: 17, XP_015615160, Os11g14950). Using the chloroplast targeting sequence prediction program ChloroP1.1, the inventors identified the putative chloroplast targeting sequence as the first 47 amino acid residues (SEQ ID NO: 18) in the rice ALS(SS) protein. To target mitochondria with the ALS(SS) protein, the inventors designed a nucleotide sequence in which the sequence encoding the first 47 aa residues in the rice ALS(SS) protein was deleted and replaced with the sequence encoding the MTS of the Arabidopsis At5g47030 gene (SEQ ID NO: 19).

[0458] The resulting DNA sequence encoding MTS-ALS(SS) is presented as Sequence ID No. 20.

[0459] The amino acid sequence of MTS-ALS(SS) encoded by SEQ ID NO: 20 is presented as SEQ ID NO: 21. The first 36 amino acids of SEQ ID NO: 21 correspond to MTS derived from the Arabidopsis At5g47030 gene.

[0460] DNA fragments encoding MTS-ALS(SS) were synthesized and operably ligated to the rice Actin1 promoter and intron (SEQ ID NO: 22) and NOS terminator (SEQ ID NO: 13) in a construct of plasmid pNAP151. Plasmid pNAP151 has an OCS terminator (SEQ ID NO: 23) at the 5' end of the MTS-ALS(SS) expression cassette. The OCS terminator and the entire MTS-ALS(SS) expression cassette derived from pNAP151 were cloned into pNAP152 to construct pNAP170. Figure 1 shows a map of plasmid pNAP170. This plasmid pNAP170 contains three nuclear expression cassettes: pUBI1::MTS-ALS(HR-LS)::OCS terminator, pACT1::MTS-ALS(SS)::NOS terminator, and p35S::HPT::CaMV terminator.

[0461] Essentially as described in Example 1, rice callus cells were transformed with pNAP170. After transforming the rice callus cells with pNAP170 using a microparticle gun, the inventors selected the event growing on a medium containing the sulfonylurea herbicide, i.e., chlorsulfuron, as the selective agent (Figure 2). Figure 2 shows the growth of rice callus cells in a medium containing chlorsulfuron, where the rice callus cells have been transformed with pNAP170. The callus within the circle was subjected to further selection.

[0462] Example 3 Sulfonylurea selection of transformed cells by expression of herbicide-resistant ALS large subunit protein in rice mitochondria The polynucleotide encoding the ALS(HR-LS) protein was introduced into rice mitochondria, and its effectiveness as a selection marker was evaluated. For this purpose, 13 of the 18 amino acids encoding the chloroplast-targeting sequence of the ALS(HR-LS) protein (described in Example 2) were deleted. Therefore, the resulting protein mALS(HR-LS) does not possess a functional organelle-targeting sequence. The amino acid sequence of the mALS(HR-LS) protein is presented as Sequence ID No. 24.

[0463] The sequence encoding mALS(HR-LS) was optimized for expression in rice mitochondria by replacing rare codons with more frequently used codons and removing unnecessary restriction sites. The optimized nucleotide sequence of the mALS(HR-LS) encoding region is presented as Sequence ID No. 25.

[0464] To express the mALS(HR-LS) gene in rice mitochondria, the sequence encoding mALS(HR-LS) was manipulatively ligated to the promoter and terminator of the ATP1 gene encoded in the rice mitochondrial genome. The ATP1 gene sequence was identified in the GenBank database (NC_011033). To enhance transcription in mitochondria, the inventors added a T7 promoter (SEQ ID NO: 26) upstream of the transcription start site. The nucleotide sequence of this hybrid ATP1+T7 promoter is presented as SEQ ID NO: 27.

[0465] Furthermore, a T7 terminator (SEQ ID NO: 28) was fused to the 5' end of an ATP1 terminator to produce a hybrid T7+ATP1 terminator (SEQ ID NO: 29) used in this experiment.

[0466] Next, a mitochondrial expression cassette for mALS(HR-LS) was cloned into pNAP76, a pBR322-based vector containing the B4 element associated with autonomous replication in rice mitochondria. Plasmid pNAP76 also encodes an eGFP reporter with an RNA editing site derived from rice COX2, which creates a translation initiation site by rice mitochondrial-specific native RNA editing (SEQ ID NO: 30). The eGFP coding sequence was operably ligated to the rice COB1 promoter and 5'UTR (SEQ ID NO: 31) and the rice COB1 terminator (SEQ ID NO: 32). The resulting construct, pNAP198, contains the mitochondrial expression cassette for mALS(HR-LS). Figure 3 shows a map of plasmid pNAP198. This plasmid contains the mALS(HR-LS) coding region operably ligated to a hybrid T7+ rice ATP1 promoter and a hybrid T7+ rice ATP1 terminator. The plasmid also contains an eGFP coding sequence that is operably ligated to the rice COB1 promoter and the 5'UTR and rice COB1 terminator. The plasmid further contains a B4 element associated with autonomous replication in rice mitochondria.

[0467] To improve sulfonylurea selection in mitochondria, the inventors constructed a nuclear expression construct of MTS-ALS(SS) and MTS-T7 RNA polymerase. This construct, pNAP195, had three expression cassettes: pUBI1::MTS-T7 Pol::OCS terminator, pACT1::MTS-ALS(SS)::NOS terminator, and p35S::HPT::CaMV terminator. The nucleotide sequence encoding MTS-T7 RNA polymerase is presented as SEQ ID NO: 33. The corresponding amino acid sequence of MTS-T7 RNA polymerase is presented as SEQ ID NO: 34. The MTS (SEQ ID NO: 35) used for MTS-T7 RNA polymerase was derived from the At5g47030 gene.

[0468] Two constructs, pNAP195 and pNAP198, were simultaneously transformed into rice callus cells, essentially as described in Example 1. After simultaneously transforming rice callus cells with pNAP195 and pNAP198 using a microparticle gun, the inventors selected the event of growth on a medium containing the sulfonylurea herbicide, i.e., chlorsulfuron, as a selective agent (Figure 4). Figure 4 shows the growth of rice callus cells in a medium containing chlorsulfuron, where the rice callus cells were simultaneously transformed with pNAP195 and pNAP198. The callus samples in the circles were subjected to further selection.

[0469] Example 4 Mitochondrial gene editing using donor DNA encoding mALS (HR-LS) In this experiment, rice mitochondria were transformed using a DNA fragment containing donor DNA encoding mALS(HR-LS). The donor DNA fragment had a region homologous to the ATP6 gene in the rice mitochondrial genome at its terminal. The homologous region was 1.6 kb at the 5' end and 1.2 kb at the 3' end. The 5' homologous region of the donor DNA is presented as SEQ ID NO: 36. Specific nucleotides were altered in the 5' homologous region to prevent recognition by gRNA2 (SEQ ID NO: 37) and the MAD7 enzyme (SEQ ID NO: 38).

[0470] The 3' homologous region of the donor DNA is presented as Sequence ID No. 39. Specific nucleotides were altered in the 3' homologous region to prevent recognition by gRNA4 (Sequence ID No. 40) and the MAD7 enzyme.

[0471] To further ensure the expression of the mALS gene in mitochondria, in two donor DNA plasmids, the inventors replaced the region encoding the start codon of mALS(HR-LS) with one of two elements derived from RNA editing sites naturally occurring in rice mitochondria: namely, the sequence encoding the RNA editing site of the rice nad4L transcript (SEQ ID NO: 41) or the sequence encoding the RNA editing site of the rice cox2 transcript (SEQ ID NO: 42).

[0472] If RNA editing originating from one of these mitochondrial-specific sites is absent, the resulting mRNA will likely lack an in-frame AUG codon near the start of the protein-coding region. Therefore, functional mALS(HR-LS) proteins are not expected to be produced, further confirming mitochondrial selection.

[0473] Each of the two mALS(HR-LS) coding regions (having alternative RNA editing sites) was operably ligated to a hybrid ATP1+T7 promoter and a cleaved form of the hybrid T7+ATP1 terminator described in Example 3 (SEQ ID NO: 43).

[0474] The nucleotide sequences of donor DNA fragments prepared using nad4L and cox2 RNA editing sites (derived from plasmids pNAP432 and pNAP433, respectively) are presented as SEQ ID NOs. 44 and 45, respectively. A map of plasmid pNAP432 is shown in Figure 5. This plasmid contains donor DNA used to transform rice mitochondria. The donor DNA has an mALS (HR-LS) coding region (containing a nad4L RNA editing site) that is manipulably linked to the cleavage forms of the hybrid T7+ rice ATP1 promoter and hybrid T7+ rice ATP1 terminator. The donor DNA has a 1.6kb rice mitochondrial DNA homology region at the 5' end and a 1.2kb region at the 3' end. Plasmid pNAP433 is identical to plasmid pNAP432 except that it has a cox2 RNA editing site instead of a nad4L RNA editing site.

[0475] Each donor DNA fragment also contained a nucleotide sequence (SEQ ID NO: 46) encoding the orf79 protein (SEQ ID NO: 47). Each donor DNA sequence also encodes a gRNA cassette (SEQ ID NO: 48) for use with the possible MAD7 nuclease.

[0476] Two donor DNA fragments were used to transform rice callus cells, either individually or with nuclear expression constructs, namely pNAP195 (for MTS-ALS(SS) expression) or pNAP159 (without MTS-ALS(SS) expression). pNAP195 is described in Example 3 and has three nuclear expression cassettes: pUBI1::MTS-T7 Pol::OCS terminator, pACT1::MTS-ALS(SS)::NOS terminator, and p35S::HPT::CaMV terminator. pNAP159 was constructed without the MTS-ALS(SS) nuclear expression cassette and has two nuclear expression cassettes: pUBI1::MTS-T7 Pol::NOS terminator and p35S::HPT::CaMV terminator.

[0477] Following transformation of rice callus cells with donor DNA fragments alone, or simultaneous transformation of donor DNA fragments with either plasmid pNAP159 or plasmid pNAP195, the inventors selected the event of growth on a medium containing chlorsulfuron, a sulfonylurea herbicide, as a selective agent (Figure 6). Figure 6A: Rice callus cells transformed simultaneously with donor DNA isolated from plasmid pNAP195 and pNAP432. Figure 6B: Rice callus cells transformed simultaneously with donor DNA isolated from plasmid pNAP195 and pNAP433.

[0478] Integrated Analysis To confirm the integration of donor DNA into the mitochondrial genome, PCR analysis of the junctional regions was performed. Each junctional region was amplified using a primer specific to the donor DNA region and another primer specific to the mitochondrial genome sequence near the homologous region of the donor DNA. The primer pair used by the inventors for amplification of the 5' junctional region was 5HR primer A (SEQ ID NO: 49) and ORF primer B (SEQ ID NO: 50). The primer pair used by the inventors for amplification of the 3' junctional region was 3HR primer A (SEQ ID NO: 51) and 420 primer A (SEQ ID NO: 52).

[0479] For PCR analysis, callus (5–20 mg) from each positive event was sampled in a tube. 300 μl of 0.02N NaOH with 1 mM EDTA was added to each sample and heated at 100°C for 20 minutes. The aqueous phase was then extracted with phenol / chloroform, followed by extraction with chloroform. Total DNA was precipitated by the addition of NaOAc and ethanol. The DNA was resuspended in 30 μl TE. The average DNA yield was approximately 200 ng / μl. 1 μl of DNA was used for each PCR reaction. The PCR reaction was prepared as follows: 1 μl of total DNA, 10 pmol of each primer, and 12.5 μl of LongAmp Taq 2X Master Mix (New England Labs Inc.) in a 25 μl reaction mixture. PCR reactions for 5' junction amplification were performed using 35 cycles of 30 seconds at 95°C, followed by 15 seconds at 95°C and 3 minutes at 65°C, and then a final incubation of 10 minutes at 65°C. PCR reactions for 3' junction amplification were performed using 35 cycles of 30 seconds at 95°C, followed by 15 seconds at 95°C, 30 seconds at 63°C, and 2 minutes at 65°C, and then a final incubation of 10 minutes at 65°C. PCR samples were separated on a 0.7% agarose gel. Junctional DNA with accurate sizes (1,742 bp at the 5' junction and 1,438 bp at the 3' junction) was amplified from multiple samples. Selected DNA bands were isolated from the gel and subjected to sequence analysis. Accurate integration of donor DNA in homologous regions was observed in all cases. Regarding the frequency of events under sulfonylurea selection, no significant difference was observed between the two RNA editing sites used by the inventors in constructs pNAP432 and pNAP433. Comparing the results from 34 events from three different combinations of mitochondrial and nuclear expression constructs (Table 2), a higher frequency of donor DNA integration was observed when combined with simultaneous T7 RNA polymerase expression. This is likely due to the potent expression of mALS(HR-LS) from T7 promoters in mitochondria containing T7 RNA polymerase. Furthermore, a higher frequency of integration was observed when combined with simultaneous ALS(SS) expression.

[0480] [Table 2]

[0481] Example 5 Selection of transformed rice cells using glyphosate or glufosinate Tissue induction, maintenance, and pre-culture in experiments utilizing glyphosate or glufosinate for the selection of transformed rice cells were the same as those described in Example 1 for callus from wild-type Nipponbare and tissue from callus events with a dexamethasone induction system.

[0482] Rice callus from both sources was transformed using a particulate bombardment method with various GS1-HR or EPSPS-HR expression constructs (e.g., ATP1 promoters driving the coding regions for herbicide-resistant (HR) forms of GS1 or EPSPS). In some experiments, the herbicide-resistant GS1-HR gene or the herbicide-resistant EPSPS-HR gene was the only selectable marker gene, while in others it was delivered along with the herbicide-resistant ALS gene and / or the oligomycin-resistant oliR gene. Following bombardment, the following steps were used for culture and selection.

[0483] 1. After bombardment, the callus was incubated in the dark at 26°C for 16–20 hours, and then the callus tissue aggregates of approximately 1–3 mM in size were subcultured in selective medium, a callus maintenance medium supplemented with either 1.5 mM glyphosate or 50–100 mg / L glufosinate ammonium (glufosinate). The tissue ...

Claims

1. A method for transforming the mitochondria of cells, a) A step of introducing a first polynucleotide encoding a first polypeptide into the mitochondria of the cell, wherein the cell is a plant cell or an algal cell, the first polypeptide encoding the first polypeptide is a variant of a naturally occurring polypeptide, the naturally occurring polypeptide contains enzyme activity inhibited by a herbicide, and the variant of the naturally occurring polypeptide contains enzyme activity that exhibits resistance to the herbicide; b) A step of growing the cells under conditions in which the first polypeptide is expressed, c) A step of growing the cells in a culture medium in which the herbicide is present at an effective concentration, d) A method comprising the step of selecting a transformed cell containing transformed mitochondria comprising the first polynucleotide.

2. A method for transforming the mitochondria of cells, a) In the mitochondria of the cell which is a plant cell or an algal cell, i) A first polynucleotide encoding a first polypeptide, wherein the first polypeptide is a variant of a naturally occurring polypeptide, the naturally occurring polypeptide contains an enzyme activity inhibited by a herbicide, and the variant of the naturally occurring polypeptide contains an enzyme activity that exhibits resistance to the herbicide, and ii) A step of introducing a second polynucleotide that codes for a selectable marker, wherein the selectable marker enables the cell to grow in the presence of a selector, and the second polynucleotide does not code for the first polypeptide, b) A step of growing the cells under conditions in which the selectable marker is expressed, c) A step of growing the cells in a culture medium containing the selector of the selectable marker, wherein the selector is present in an effective concentration; d) A method comprising the step of selecting transformed cells containing transformed mitochondria, wherein the transformed mitochondria contain the first polynucleotide, and further, the transformed cells containing the transformed mitochondria can be grown in a culture medium containing the herbicide, wherein the herbicide is present at an effective concentration.

3. The method according to claim 2, wherein the selective agent is toxic to the cells.

4. The method according to claim 2, wherein the selectable marker encoded by the second polynucleotide is a phosphyte dehydrogenase enzyme or a biologically active fragment thereof, and the selector is a phosphyte.

5. The method according to claim 2, wherein the selectable marker encoded by the second polynucleotide is oligomycin-resistant ATP6 or a biologically active fragment thereof, the selector is oligomycin, and the culture medium containing the selector optionally contains disulfiram.

6. The method according to any one of claims 1 to 5, wherein the transformed mitochondria comprises an edited mitochondrial genome containing the first polynucleotide.

7. The method according to any one of claims 1 to 6, wherein the naturally occurring polypeptide is present in the cytosol, the plastid, or both.

8. The method according to claim 7, wherein the naturally occurring polypeptide is encoded by a nuclear gene.

9. The method according to claim 8, wherein the enzyme activity of the naturally occurring polypeptide comprises at least one selected from the group consisting of acetolactic acid synthase (ALS) activity, 5-enol-pyruvir-sikimate-3-phosphate synthase (EPPSPS) activity, glutamine synthase (GS) activity, and any combination thereof.

10. The method according to claim 9, wherein the enzyme activity of the naturally occurring polypeptide is acetolactate synthase activity, and the herbicide is an acetolactate synthase inhibitor.

11. The method according to claim 10, wherein the first polypeptide comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

24.

12. The method according to claim 11, wherein the amino acid sequence of the first polypeptide includes or consists of SEQ ID NO:

24.

13. The method according to claim 10, wherein the first polynucleotide encoding the first polypeptide has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

25.

14. The method according to claim 13, wherein the first polynucleotide includes or consists of SEQ ID NO:

25.

15. The method according to any one of claims 10 to 14, wherein the acetolactate synthase inhibitor comprises sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl benzoate, sulfonanilide, sulfonyl-aminocarbonyl-triazolinone, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

16. The method according to claim 15, wherein the sulfonylurea comprises chlorsulfuron.

17. The method according to claim 16, wherein the culture medium containing the herbicide contains chlorsulfuron at a concentration of 20 nM to 100 nM, 100 nM to 1 μM, 1 μM to 20 μM, or 20 μM to 100 μM.

18. The method according to claim 15, wherein the imidazolinone comprises imazapyr, imazapic, imazetapyr, imazamox, imazametabenz, imazakine, any salt thereof, any stereoisomer thereof, or any combination thereof.

19. The method according to claim 15, wherein the triazolopyrimidine comprises penoxysram, chloransram-methyl, diclosram, florasram, flumethosram, metosram, pyroxysram, any salt thereof, any stereoisomer thereof, or any combination thereof.

20. The method according to claim 15, wherein the pyrimidinyl benzoate comprises bispyribac sodium, pyribenzoxime, pyrithiobac sodium, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

21. The method according to claim 15, wherein the sulfonanilide comprises pyrimisulfan, triafamone, a salt of any of these, a stereoisomer of any of these, or any combination thereof.

22. The method according to claim 15, wherein the sulfonylaminocarbonyltriazolinone comprises fulcarbazone-sodium, propoxycarbazone-sodium, thiencarbazone-methyl, any salt thereof, any stereoisomer thereof, or any combination thereof.

23. The aforementioned sulfonylurea includes amidesulfuron, azimsulfuron, bensulfuron-methyl, chlorimulon-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, etamethosulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron-methyl-na, horamsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron-methyl-na, mesosulfuron-methyl, metazosulfuron, metosulfuron-methyl, and nicosulfuron. The method according to claim 15, comprising orthosulfamurone, oxasulfurone, primisulfurone-methyl, propyrisulfurone, prosulfurone, pyrazosulfurone-ethyl, limsulfurone, sulfomethurone-methyl, sulfosulfurone, thifensulfurone-methyl, triasulfurone, tribenulon-methyl, trifloxysulfurone-na, triflusulfurone-methyl, tritosulfurone, any salt of any of these, any stereoisomer of any of these, or any combination thereof.

24. A step of introducing a mALS-SS polynucleotide encoding a regulatory subunit of acetolactate synthase or a biologically active fragment thereof into the mitochondria, The steps include growing the cells under conditions in which the regulatory subunit of acetolactic acid synthase or a biologically active fragment thereof is expressed, and The method according to any one of claims 9 to 23, further comprising:

25. A step of introducing an nMTS-ALS-SS polynucleotide encoding a modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof into the nucleus of a cell, wherein the modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof comprises the regulatory subunit of acetolactate synthase or a biologically active fragment thereof operably linked to a mitochondrial-targeting peptide, The steps include growing the cells under conditions in which the modified regulatory subunit of acetolactic acid synthase or the modified biologically active fragment thereof is expressed, and The method according to any one of claims 9 to 24, further comprising:

26. The method according to claim 25, wherein the modified regulatory subunit of acetolactate synthase or the modified biologically active fragment comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:

21.

27. The method according to claim 24, further comprising the step of selecting transformed cells, wherein the transformed mitochondria contain the mALS-SS polynucleotide.

28. The method according to claim 25 or 26, further comprising the step of selecting transformed cells containing transformed nuclei, wherein the transformed nuclei contain the nMTS-ALS-SS polynucleotide.

29. The method according to claim 9, wherein the enzyme activity of the naturally occurring polypeptide is EPSPS activity, and the herbicide is an EPSPS inhibitor.

30. The method according to claim 29, wherein the first polypeptide comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

62.

31. The method according to claim 30, wherein the amino acid sequence of the first polypeptide includes or consists of SEQ ID NO:

62.

32. The method according to claim 29, wherein the first polynucleotide encoding the first polypeptide has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

63.

33. The method according to claim 32, wherein the first polynucleotide includes or consists of SEQ ID NO:

63.

34. The method according to any one of claims 29 to 33, wherein the herbicide comprises glyphosate.

35. The method according to claim 34, wherein the culture medium containing the herbicide contains glyphosate at a concentration of at least 1.5 mM.

36. The method according to claim 9, wherein the enzyme activity of the naturally occurring polypeptide is glutamine synthetase activity, and the herbicide is a glutamine synthetase inhibitor.

37. The method according to claim 36, wherein the first polypeptide comprises an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

72.

38. The method according to claim 37, wherein the amino acid sequence of the first polypeptide includes or consists of SEQ ID NO:

72.

39. The method according to claim 36, wherein the first polynucleotide encoding the first polypeptide has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

73.

40. The method according to claim 39, wherein the first polynucleotide includes or consists of Sequence ID No.

73.

41. The method according to any one of claims 36 to 40, wherein the herbicide comprises at least one selected from the group consisting of glufosinate, bialaphos, fosalacin, and any combination thereof.

42. The method according to claim 41, wherein the herbicide comprises the glufosinate.

43. The method according to claim 42, wherein the culture medium containing the herbicide contains glufosinate at a concentration of 50 to 100 mg / L.

44. The method according to any one of claims 1 to 43, wherein the cells are plant cells selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, cotton cells, and soybean cells.

45. A step of introducing donor DNA into the mitochondria of the cell, wherein the donor DNA is directed in the 5'-3' direction, a) 5' homologous region (HR) polynucleotide, b) Internal polynucleotides that are heterogeneous to the mitochondria, and c) 3'-HR polynucleotide The process includes a step in which the 5'-HR polynucleotide and the 3'-HR polynucleotide each contain sequences capable of homologous recombination with an endogenous mitochondrial DNA sequence, and homologous recombination between all or part of the donor DNA and the endogenous mitochondrial DNA sequence incorporates all or part of the internal polynucleotides into the endogenous mitochondrial DNA sequence, A step of selecting transformed cells having an edited mitochondrial genome, wherein the edited mitochondrial genome includes all or part of the internal polynucleotides. The method according to any one of claims 1 to 44, further comprising:

46. The method according to claim 45, wherein the donor DNA comprises both the first polynucleotide and the second polynucleotide encoding the first polynucleotide and a selectable marker.

47. The method according to claim 46, wherein the edited mitochondrial genome comprises the first polynucleotide.

48. The method according to claim 46 or 47, wherein the edited mitochondrial genome comprises the second polynucleotide encoding the selectable marker.

49. The method according to claim 45, wherein the donor DNA does not contain the first polynucleotide.

50. The method according to claim 49, wherein the donor DNA does not contain the second polynucleotide encoding a selectable marker.

51. The method according to any one of claims 45 to 50, wherein the internal polynucleotide of the donor DNA encodes a polypeptide, a functional RNA, or both.

52. The method according to any one of claims 45 to 51, wherein the internal polynucleotides of the donor DNA include a cytoplasmic male sterility (CMS) coding region.

53. The method according to claim 52, wherein the CMS code area includes rice orf79.

54. The method according to claim 52, wherein the CMS code region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to sequence number 47.

55. The method according to claim 54, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

47.

56. The method according to any one of claims 53 to 55, wherein the cells are rice cells.

57. The method according to claim 52, wherein the CMS code area includes wheat orf256 or wheat orf279.

58. The method according to claim 52, wherein the CMS code region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to sequence number 54.

59. The method according to claim 58, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

54.

60. The method according to claim 52, wherein the CMS code region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to sequence number 56.

61. The method according to claim 60, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

56.

62. The method according to any one of claims 57 to 61, wherein the cells are wheat cells.

63. The method according to any one of claims 45 to 62, wherein the homologous recombination sequence in the 5'-HR polynucleotide has a size of 25 to 75 nucleotides, 25 to 100 nucleotides, 25 to 150 nucleotides, 25 to 200 nucleotides, 25 to 300 nucleotides, 25 to 400 nucleotides, 25 to 500 nucleotides, 25 to 1000 nucleotides, 25 to 1500 nucleotides, or 25 to 2000 nucleotides.

64. The method according to any one of claims 45 to 63, wherein the homologous recombination sequence in the 3'-HR polynucleotide has a size of 25 to 75 nucleotides, 25 to 100 nucleotides, 25 to 150 nucleotides, 25 to 200 nucleotides, 25 to 300 nucleotides, 25 to 400 nucleotides, 25 to 500 nucleotides, 25 to 1000 nucleotides, 25 to 1500 nucleotides, or 25 to 2000 nucleotides.

65. The method according to any one of claims 45 to 64, wherein at least one selected from the group consisting of the first polynucleotide, the second polynucleotide encoding a selectable marker, the internal polynucleotide, the 5'-HR polynucleotide, the 3'-HR polynucleotide, and any combination thereof is introduced into the cells via microinjection, meristematic tissue transformation, electroporation, Agrobacterium-mediated transformation, virus-based gene transfer, transfection, vacuum infiltration, particulate gun bombardment, or any combination thereof.

66. The method according to any one of claims 45 to 65, wherein at least one selected from the group consisting of the first polynucleotide, the second polynucleotide encoding a selectable marker polynucleotide, the internal polynucleotide, the 5'-HR polynucleotide, the 3'-HR polynucleotide, and any combination thereof is introduced into the cell as a carbon nanotube-polynucleotide complex or as a peptide-polynucleotide complex, the peptide-polynucleotide complex comprising at least one peptide.

67. The method according to claim 66, wherein the at least one peptide of the peptide-polynucleotide complex comprises at least one selected from the group consisting of cell-permeable peptides (CPPs), organelle-targeting peptides, mitochondrial-targeting peptides, histidine-rich peptides, lysine-rich peptides, and any combination thereof.

68. a) In the mitochondria of the cell, i) an mRNA polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA guides the polynucleotide-derived polypeptide to cleave at least one target sequence present in the organelle genome, ii) an mPGP polynucleotide encoding the polynucleotide-derived polypeptide, wherein the polynucleotide-derived polypeptide is associated with at least one guide RNA, and the mPGP polynucleotide cleaves the at least one target sequence. The method according to any one of claims 1 to 67, further comprising the step of introducing a recombinant DNA construct containing the above.

69. a) In the nucleus of the cell, i) nMTS-PGP polynucleotides encoding a modified polynucleotide-derived polypeptide, wherein the modified polynucleotide-derived polypeptide comprises a polynucleotide-derived polypeptide operably linked to a mitochondrial targeting peptide, and the polynucleotide-derived polypeptide, when associated with at least one guide RNA, cleaves at least one target sequence present in the mitochondrial genome, and ii) An nGRNA polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA guides the polynucleotide-derivable polypeptide to cleave at least one target sequence present in the mitochondrial genome. The method according to any one of claims 1 to 67, further comprising the step of introducing a.

70. a) In the nucleus of the cell, i) A step of introducing an nMTS-PGP polynucleotide that cleaves at least one target sequence present in the mitochondrial genome, wherein the nMTS-PGP polynucleotide encodes a modified polynucleotide-derived polypeptide, the modified polynucleotide-derived polypeptide comprises a polynucleotide-derived polypeptide operably linked to a mitochondrial targeting peptide, and the polynucleotide-derived polypeptide is associated with at least one guide RNA. b) In the mitochondria of the cell, i) A step of introducing an mRNA polynucleotide which encodes at least one guide RNA, wherein the at least one guide RNA guides the polynucleotide-derivable polypeptide to cleave at least one target sequence present in the mitochondrial genome. The method according to any one of claims 1 to 67, further comprising:

71. The method according to any one of claims 68 to 70, wherein the polynucleotide-derived polypeptide is at least one selected from the group consisting of Cas9 protein, Cas3 protein, MAD2 protein, MAD7 protein, CRISPR nuclease, the nuclease domain of Cas protein, Cpf1 protein, Argonaut, modified forms thereof, biologically active fragments thereof, and any combination thereof.

72. The method according to any one of claims 68 to 71, wherein homologous recombination of all or part of donor DNA with an endogenous mitochondrial DNA sequence results in an edited mitochondrial genome lacking at least one target sequence.

73. The method according to any one of claims 1 to 72, further comprising the step of introducing an nMTS-SDN polynucleotide into the nucleus of a cell, wherein the nMTS-SDN polynucleotide encodes a modified site-specific nuclease, the modified site-specific nuclease comprises a site-specific nuclease operably linked to a mitochondrial-targeting peptide, and the site-specific nuclease cleaves at least one site-specific target sequence present in the mitochondrial genome.

74. The method according to claim 73, wherein the site-specific nuclease is at least one selected from the group consisting of TALEN, zinc finger nuclease, meganuclease, restriction enzyme, and any combination thereof.

75. a) In the nucleus of the cell, A step of introducing an nMTS-Rep polynucleotide encoding a modified Rep protein, which includes a Rep protein functionally linked to a mitochondrial-targeting peptide, b) In the mitochondria of the cell, VOR-donor-VOR polynucleotide, in the 5'-3' direction, i) VOR array, ii) Donor DNA, and iii) Second VOR sequence The method according to any one of claims 45 to 74, further comprising the step of introducing a VOR-donor-VOR polynucleotide containing

76. The method according to claim 75, wherein the cells are grown under conditions in which the modified Rep protein is expressed.

77. The method according to claim 75 or 76, wherein the Rep protein and the VOR sequence are derived from a geminivirus.

78. The method according to claim 77, wherein the modified Rep protein includes or consists of SEQ ID NO: 111 or a nucleic acid sequence encoding the modified Rep protein, and the VOR sequence includes or consists of SEQ ID NO:

71.

79. The method according to any one of claims 75 to 78, wherein the donor DNA is introduced into the mitochondria of the cell and then amplified, and the expression level of the donor DNA transformed with the nMTS-Rep polynucleotide, the VOR sequence, and the second VOR sequence is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than the expression level of the donor DNA transformed without the nMTS-Rep polynucleotide, the VOR sequence, or the second VOR sequence.

80. The method according to any one of claims 75 to 79, wherein the nMTS-Rep polynucleotide encoding the modified Rep protein is operably linked to an inducible promoter.

81. The method according to claim 80, wherein the inductive promoter is induced by dexamethasone.

82. The method according to any one of claims 1 to 81, wherein the first polynucleotide encoding the first polypeptide further comprises a T7 RNA polymerase promoter, and the expression of the first polypeptide is under the control of the T7 RNA polymerase promoter.

83. The method according to claim 82, further comprising the step of introducing an MTS-T7P polynucleotide encoding a modified T7 RNA polymerase into the nucleus of the cell, wherein the modified T7 RNA polymerase comprises a T7 RNA polymerase operably linked to a mitochondrial-targeting peptide.

84. The method according to any one of claims 1 to 83, wherein the sequence encoding the start codon of the first polypeptide is replaced with a sequence encoding a mitochondrial RNA editing site.

85. The method according to claim 84, wherein the mitochondrial RNA editing site is at least one selected from the group consisting of the rice mitochondrial nad4L gene, the rice mitochondrial cox2 gene, the wheat mitochondrial cox2 gene, and any combination thereof.

86. The method according to claim 85, wherein the sequence encoding the mitochondrial RNA editing site includes SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO:

80.

87. The method according to any one of claims 1 to 86, further comprising the steps of introducing a third polynucleotide encoding an additional selectable marker polypeptide into the nucleus of the cell, wherein the additional selectable marker polypeptide confers resistance to an additional selector, and selecting cells that grow in the presence of the additional selector.

88. The method according to claim 87, wherein the cells grow simultaneously in the presence of the additional selective agent and the herbicide, and the additional selective agent and the herbicide are present at effective concentrations, respectively.

89. The method according to claim 87, wherein the cells grow continuously, first in the presence of the additional selector and then in the presence of the herbicide, or grow continuously, first in the presence of the herbicide and then in the presence of the additional selector, wherein the additional selector and the herbicide are present at effective concentrations.

90. The method according to any one of claims 87 to 89, wherein the additional selectable marker polypeptide is a polypeptide having hygromycin phosphotransferase (HPT) activity, and the additional selector is hygromycin.

91. The method according to any one of claims 45 to 90, further comprising the step of removing the first polynucleotide encoding the first polypeptide from the transformed mitochondria after the incorporation of the internal polynucleotide of the donor DNA.

92. The method according to any one of claims 6 to 91, further comprising the step of selecting cells containing a plurality of mitochondrial genomes, wherein at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the plurality of mitochondrial genomes contain edited mitochondrial genomes.

93. The method according to any one of claims 6 to 92, further comprising the step of selecting cells that are isocytoplasmic to the edited mitochondrial genome.

94. The method according to any one of claims 1 to 93, wherein the cells are plant cells, and a plant grows from the plant cells.

95. The method according to claim 94, further comprising the step of selecting a plant containing the first polypeptide.

96. Cells produced by the method according to any one of claims 1 to 95, which are plant cells selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, cotton cells, and soybean cells.

97. Plants, cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, produced from plant cells according to claim 96, wherein the cells, tissues, reproductive materials, seeds, roots, leaves, flowers, fruits, pollen, offspring, or parts thereof, or any combination thereof, include an edited mitochondrial genome.

98. A method for eradicating weeds, A method comprising the step of growing a plurality of plants in the presence of a herbicide that is a plant enzyme inhibitor, wherein at least one of the plurality of plants contains mitochondria containing a heterologous polynucleotide encoding a variant of the plant enzyme, the variant of the plant enzyme has enzymatic activity that exhibits resistance to the herbicide, the presence of the herbicide is sufficient to selectively promote the growth of the at least one of the plurality of plants, and the growth of the at least one of the plurality of plants is increased compared to plants lacking the heterologous polynucleotide.

99. The method according to claim 98, further comprising the step of applying the herbicide to the at least one plant, the plurality of plants, the soil adjacent to the at least one plant, the soil adjacent to the plurality of plants, or any combination thereof.

100. The method according to claim 99, wherein the herbicide is applied as a leaf conditioner.

101. The method according to claim 99, wherein the herbicide is applied as a soil conditioner.

102. The method according to any one of claims 98 to 101, wherein at least one of the plurality of plants is selected from the group consisting of wheat, corn, rice, barley, sorghum, rye, sugarcane, potato, tomato, canola, broccoli, cauliflower, cotton, and soybean.

103. The method according to any one of claims 98 to 102, wherein the plant lacking the heterologous polynucleotide is a weed.

104. The method according to any one of claims 98 to 103, wherein the plant enzyme is at least one selected from the group consisting of acetolactic acid synthase (ALS), 5-enol-pyruvir-sikimate-3-phosphate synthase (EPPSPS), glutamine synthase (GS), and any combination thereof.

105. The method according to claim 104, wherein the plant enzyme is acetolactate synthase, and the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

24.

106. The method according to claim 105, wherein the amino acid sequence of the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide includes or consists of Sequence ID No.

24.

107. The method according to claim 104, wherein the plant enzyme is 5-enol-pyruvir-sikimate-3-phosphate synthase (EPPSPS), and the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

62.

108. The method according to claim 107, wherein the amino acid sequence of the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide includes or consists of Sequence ID No.

62.

109. The method according to claim 104, wherein the plant enzyme is glutamine synthetase, and the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

72.

110. The method according to claim 109, wherein the amino acid sequence of the mutant of the plant enzyme having the enzyme activity that exhibits resistance to the herbicide includes or consists of SEQ ID NO:

72.

111. A cell comprising an edited mitochondrial genome, wherein the cell is a plant cell or an algal cell, and the edited mitochondrial genome comprises heterologous polynucleotides encoding a variant of a naturally occurring polypeptide having enzymatic activity, the enzymatic activity of the naturally occurring polypeptide being inhibited by a herbicide, and the enzymatic activity of the variant of the naturally occurring polypeptide having enzymatic activity being resistant to the herbicide.

112. The cell according to claim 111, which is a plant cell selected from the group consisting of wheat cells, maize cells, rice cells, barley cells, sorghum cells, rye cells, canola cells, broccoli cells, cauliflower cells, cotton cells, and soybean cells.

113. The cell according to claim 111 or 112, wherein the edited mitochondrial genome comprises at least one nucleotide substitution, deletion, insertion, or any combination thereof.

114. The cell according to any one of claims 111 to 113, wherein the cell comprises transformed mitochondria containing the edited mitochondrial genome.

115. The cell according to any one of claims 111 to 114, wherein the enzyme activity of the naturally occurring polypeptide is at least one selected from the group consisting of acetolactic acid synthase (ALS) activity, 5-enol-pyruvir-sikimate-3-phosphate synthase (EPPSPS) activity, glutamine synthase (GS) activity, and any combination thereof.

116. The cell according to claim 115, wherein the enzymatic activity of the naturally occurring polypeptide is acetolactate synthase activity, and the variant of the naturally occurring polypeptide contains an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

24.

117. The cell according to claim 116, wherein the amino acid sequence of the mutant includes or consists of SEQ ID NO:

24.

118. The cell according to claim 115, wherein the enzymatic activity of the naturally occurring polypeptide is 5-enol-pyruvir-sikimate-3-phosphate synthase (EPPSPS) activity, and the variant of the naturally occurring polypeptide contains an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

62.

119. The cell according to claim 118, wherein the amino acid sequence of the variant of the naturally occurring polypeptide includes or consists of SEQ ID NO:

62.

120. The cell according to claim 115, wherein the enzymatic activity of the naturally occurring polypeptide is glutamine synthetase activity, and the variant of the naturally occurring polypeptide contains an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

72.

121. The cell according to claim 120, wherein the amino acid sequence of the variant of the naturally occurring polypeptide includes or consists of SEQ ID NO:

72.

122. The cell according to any one of claims 111 to 121, wherein the sequence encoding the start codon of the heterologous polynucleotide is replaced with the sequence encoding the mitochondrial RNA editing site.

123. The cell according to claim 122, wherein the mitochondrial RNA editing site is at least one selected from the group consisting of the rice mitochondrial nad4L gene, the rice mitochondrial cox2 gene, the wheat mitochondrial cox2 gene, and any combination thereof.

124. The cell according to claim 123, wherein the sequence encoding the mitochondrial RNA editing site includes SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO:

80.

125. The cell according to any one of claims 111 to 124, wherein the edited mitochondrial genome further comprises a second polynucleotide encoding a polypeptide or a functional RNA, or both, and the polypeptide and the functional RNA are heterogeneous with respect to the mitochondria.

126. The cell according to claim 125, wherein the second polynucleotide comprises a cytoplasmic male sterility (CMS) coding region.

127. The cell according to claim 126, wherein the CMS code region is rice orf79.

128. The cell according to claim 127, wherein the CMS coding region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

47.

129. The cell according to claim 128, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

47.

130. The cell according to any one of claims 126 to 129, wherein the cell is a rice cell.

131. The cell according to claim 126, wherein the CMS code region is wheat orf256 or wheat orf279.

132. The cell according to claim 131, wherein the CMS coding region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

54.

133. The cell according to claim 132, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

54.

134. The cell according to claim 131, wherein the CMS coding region encodes a polypeptide comprising an amino acid sequence having at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:

56.

135. The cell according to claim 134, wherein the amino acid sequence of the polypeptide includes or consists of SEQ ID NO:

56.

136. The cell according to any one of claims 131 to 135, wherein the cell is a wheat cell.

137. A cell according to any one of claims 111 to 136, comprising a plurality of mitochondrial genomes, wherein at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the plurality of mitochondrial genomes comprises the edited mitochondrial genome.

138. The cell according to any one of claims 111 to 136, wherein the cytoplasm is isoplasmic to the edited mitochondrial genome.

139. A cell according to any one of claims 111 to 138, which expresses the variant of the naturally occurring polypeptide.

140. The cell according to any one of claims 115 to 117, wherein the edited mitochondrial genome comprises heterologous mALS-SS polynucleotides encoding a regulatory subunit of acetolactate synthase or a biologically active fragment thereof.

141. The cell according to claim 140, expressing the regulatory subunit of acetolactic acid synthase encoded by a heterologous polynucleotide of mALS-SS or a biologically active fragment thereof.

142. The cell according to any one of claims 115 to 117, wherein the nucleus of the cell comprises a heterologous nMTS-ALS-SS polynucleotide encoding a modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof, and the modified regulatory subunit of acetolactate synthase or a modified biologically active fragment thereof comprises the regulatory subunit of acetolactate synthase or a biologically active fragment thereof operably linked to a mitochondrial-targeting peptide.

143. The cell according to claim 142, expressing the modified regulatory subunit of acetolactate synthase or the modified biologically active fragment thereof.

144. A cell according to any one of claims 111 to 143, which grows in a culture medium containing the herbicide present at an effective concentration.

145. A transgenic plant or a portion thereof comprising the cells described in any one of claims 111 to 144.

146. The transgenic plant or part thereof according to claim 145, further comprising cells, tissues, reproductive material, seeds, pollen, offspring, or any combination thereof.

147. A transgenic plant or part thereof according to claim 145 or 146, grown in a temperature-controlled incubator.

148. The transgenic plant or portion thereof according to claim 147, wherein the temperature-controlled incubator further includes a light-dark cycle.

149. A field or greenhouse comprising the transgenic plant or a part thereof as described in claim 145.

150. A food comprising the cells described in any one of claims 111 to 144.

151. A field containing the cells described in any one of claims 111 to 144.

152. A kit comprising the cells described in any one of claims 111 to 144 or the transgenic plant or a portion thereof described in any one of claims 145 to 148.

Images