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Definitions

• a Cmsl protein has at least about 80% identity with a sequence selected from the group consisting of SEQ ID NOs: 16-19, 24-27, 70-146, 174- 176, 212-215, and 255-287.
• the DNA constructs comprising polynucleotide sequences that encode the Cmsl proteins of the invention, or the Cmsl proteins of the invention themselves, can be used to direct the modification of genomic DNA at pre-determined genomic loci. Methods to use these DNA constructs to modify genomic DNA sequences are described herein. Modified eukaryotes and eukaryotic cells, including yeast, amoebae, insects, fungi, mammals, plants, plant cells, plant parts and seeds as well as modified prokaryotes, including bacteria and archaea, are also
• Cmsl polypeptides can be wild type Cmsl polypeptides, modified Cmsl polypeptides, or a fragment of a wild type or modified Cmsl polypeptide.
• the Cmsl polypeptide can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
• nuclease i.e., DNase, RNase
• the Cmsl polypeptide can be truncated to remove domains that are not essential for the function of the protein.
• the Cmsl polypeptides disclosed herein can further comprise at least one nuclear localization signal (NLS).
• NLS nuclear localization signal
• an NLS comprises a stretch of basic amino acids. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101- 5105).
• the NLS can be located at the N-terminus, the C-terminus, or in an internal location of the Cmsl polypeptide.
• the Cmsl polypeptide can further comprise at least one cell-penetrating domain.
• the cell-penetrating domain can be located at the N-terminus, the C- terminus, or in an internal location of the protein.
• the plastid, mitochondrial, or dual-targeting signal peptide can be located at the N-terminus, the C-terminus, or in an internal location of the Cmsl polypeptide.
• sequences metagenomically-derived sequences whose native host organism is unclear or unknown.
• methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology or identity to the sequences set forth herein.
• Sequences isolated based on their sequence identity to the entire Cmsl sequences set forth herein or to variants and fragments thereof are encompassed by the present invention.
• Such sequences include sequences that are orthologs of the disclosed Cmsl sequences. "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation.
• Fusion proteins are provided herein comprising a Cmsl polypeptide, or a fragment or variant thereof, and an effector domain.
• the Cmsl polypeptide can be directed to a target site by a guide RNA, at which site the effector domain can modify or effect the targeted nucleic acid sequence.
• the effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
• a Cmsl polypeptide can have a mutation in a position corresponding to positions 701 or 922 of SmCmsl (SEQ ID NO: 10) or to positions 848 and 1213 of SulfCmsl (SEQ ID NO: 11) when aligned for maximum identity.
• the nuclease domain can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using known methods, such as site-directed mutagenesis, PCR- mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
• the Cmsl polypeptide of the fusion protein is modified by mutating the RuvC-like domain such that the Cmsl polypeptide has no nuclease activity.
• the heterodimer can also comprise one fusion protein and an additional protein.
• the additional protein can be a nuclease.
• the nuclease is a zinc finger nuclease.
• a zinc finger nuclease comprises a zinc finger DNA binding domain and a cleavage domain.
• a zinc finger recognizes and binds three (3) nucleotides.
• a zinc finger DNA binding domain can comprise from about three zinc fingers to about seven zinc fingers.
• the zinc finger DNA binding domain can be derived from a naturally occurring protein or it can be engineered. See, for example, Beerli et al. (2002) Nat. Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem.
• inducible promoters examples include the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the In2-2 promoter which is safener induced (U.S. Pat. No.
• TR1' gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics.
• Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;
• the vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in “Current Protocols in Molecular Biology” Ausubel et al, John Wiley & Sons, New York, 2003 or "Molecular Cloning: A
• the guide RNA comprises a single molecule comprising all three regions. In other embodiments, the guide RNA can comprise two separate molecules.
• the first RNA molecule can comprise the first region of the guide RNA and one half of the "stem" of the second region of the guide RNA.
• the second RNA molecule can comprise the other half of the guide RNA
• the upstream and downstream sequences in the donor polynucleotide can have about 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% sequence identity with sequence upstream or downstream to the targeted site.
• the upstream and downstream sequences in the donor polynucleotide can have about 95% or 100% sequence identity with nucleotide sequences upstream or downstream to the targeted site.
• the donor polynucleotide can additionally comprise at least one targeted cleavage site that is recognized by the Cmsl polypeptide.
• the targeted cleavage site added to the donor polynucleotide can be placed upstream or downstream or both upstream and downstream of the donor sequence.
• the donor sequence can be flanked by targeted cleavage sites such that, upon cleavage by the Cmsl polypeptide, the donor sequence is flanked by overhangs that are compatible with those in the nucleotide sequence generated upon cleavage by the Cmsl polypeptide. Accordingly, the donor sequence can be ligated with the cleaved nucleotide sequence during repair of the double stranded break by a non-homologous repair process.
• the donor polynucleotide will be DNA.
• the DNA may be single- stranded or double-stranded and/or linear or circular.
• the donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
• the donor polynucleotide comprising the donor sequence can be part of a plasmid vector. In any of these situations, the donor polynucleotide comprising the donor sequence can further comprise at least one additional sequence.
• a construct which has no known effect on the trait of interest such as a construct comprising a marker gene
• a construct comprising a marker gene a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene
• a plant or plant cell which is a non- transformed segregant among progeny of a subject plant or plant cell
• a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
• coding sequences can be made using the methods disclosed herein to increase the level of preselected amino acids in the encoded polypeptide.
• the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S.
• Sterility genes can also be modified and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Patent No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development. Additional sterility traits are described for example in U.S. Patent Application 2016/0208243, herein incorporated by reference.
• the methods disclosed herein can also be used for insertion of heterologous genes and/or modification of native plant gene expression to achieve desirable plant traits.
• Such traits include, for example, disease resistance, herbicide tolerance, drought tolerance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content.
• Genes capable of conferring these desirable traits are disclosed in U.S. Patent Application 2016/0208243, herein incorporated by reference.
• the donor sequence can be ligated directly with the cleaved nucleotide sequence by a non-homologous repair process during repair of the double- stranded break.
• Exchange or integration of the donor sequence into the nucleotide sequence modifies the targeted nucleotide sequence or introduces an exogenous sequence into the targeted nucleotide sequence of the non-plant eukaryotic cell or organelle.
• the targeted nucleotide sequence can be modified or inactivated.
• a single nucleotide change can give rise to an altered protein product, or a shift in the reading frame of a coding sequence can inactivate or "knock out" the sequence such that no protein product is made.
• the donor sequence can be ligated directly with the cleaved nucleotide sequence by a non-homologous repair process during repair of the double- stranded break.
• Exchange or integration of the donor sequence into the nucleotide sequence modifies the targeted nucleotide sequence or introduces an exogenous sequence into the targeted nucleotide sequence of the prokaryotic cellular DNA.
• the double-stranded breaks caused by the action of the Cmsl nuclease or nucleases are repaired in such a way that DNA is deleted from the prokaryotic cellular DNA.
• polypeptide, and optional donor polynucleotide are incubated with a viral DNA sequence of interest outside of a cellular host.
• the donor sequence in embodiments in which the donor sequence is flanked by compatible overhangs (or the compatible overhangs are generated in situ by the Cmsl polypeptide) can be ligated directly with the cleaved targeted DNA sequence by a non-homologous repair process during repair of the double-stranded break. Exchange or integration of the donor sequence into the targeted DNA sequence modifies the targeted DNA sequence or introduces an exogenous sequence into the targeted DNA sequence.
• the resultant double- stranded breaks can be repaired by a non- homologous repair process such that deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break.
• transcriptional repressor domain activates or represses expression, respectively, of a gene or genes located near the targeted DNA sequence. That is, transcription may be affected for genes in close proximity to the targeted DNA sequence or may be affected for genes located at further distance from the targeted DNA sequence. It is well-known in the art that gene transcription can be regulated by distantly located sequences that may be located thousands of bases away from the transcription start site or even on a separate chromosome (Harmston and Lenhard (2013) Nucleic Acids Res 41:7185-7199).
• the modified chromosomal sequence can also be altered such that it codes for a variant protein product.
• a genetically modified eukaryote comprising a modified chromosomal sequence can comprise a targeted point mutation(s) or other modification such that an altered protein product is produced.
• the chromosomal sequence can be modified such that at least one nucleotide is changed and the expressed protein comprises one changed amino acid residue (missense mutation).
• the chromosomal sequence can be modified to comprise more than one missense mutation such that more than one amino acid is changed.
• the chromosomal sequence can be modified to have a three nucleotide deletion or insertion such that the expressed protein comprises a single amino acid deletion or insertion.
• the altered or variant protein can have altered properties or activities compared to the wild type protein, such as altered substrate specificity, altered enzyme activity, altered kinetic rates, etc.
• a Cre-lox recombination system comprises a Cre recombinase enzyme, a site-specific DNA recombinase that can catalyze the recombination of a nucleic acid sequence between specific sites (lox sites) in a nucleic acid molecule. Methods of using this system to produce temporal and tissue specific expression are known in the art.
• the modified DNA sequence of the viruses and viral genomes may be modified such that it is inactivated, has up-regulated or down-regulated expression, or produces an altered protein product, or comprises an integrated sequence.
• the modified DNA sequence may be inactivated such that the sequence is not transcribed and/or a functional protein product is not produced.
• DNA-targeting RNA or a DNA polynucleotide encoding a DNA-targeting RNA
• the DNA-targeting RNA comprises: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA; and (b) a second segment that interacts with a Cmsl polypeptide; and
• a Cmsl polypeptide or a polynucleotide encoding a Cmsl polypeptide, wherein the Cmsl polypeptide comprises: (a) an RNA-binding portion that interacts with the DNA- targeting RNA; and (b) an activity portion that exhibits site-directed enzymatic activity.
• nucleotide sequence at a target site in the genome of a plant cell encodes an SBPase, FBPase, FBP aldolase, AGPase large subunit, AGPase small subunit, sucrose phosphate synthase, starch synthase, PEP carboxylase, pyruvate phosphate dikinase, transketolase, rubisco small subunit, or rubisco activase protein, or encodes a transcription factor that regulates the expression of one or more genes encoding an SBPase, FBPase, FBP aldolase, AGPase large subunit, AGPase small subunit, sucrose phosphate synthase, starch synthase, PEP carboxylase, pyruvate phosphate dikinase, transketolase, rubisco small subunit, or rubisco activase protein.
• nuclear localization signal comprises SEQ ID NO: l, or is encoded by SEQ ID NO:2.
• a nucleic acid molecule comprising a polynucleotide sequence encoding a Cmsl polypeptide, wherein said polynucleotide sequence has been codon optimized for expression in a prokaryotic cell, wherein said prokaryotic cell is not the natural host of said Cmsl polypeptide.
• nucleic acid molecule of embodiment 51 wherein said mutated Cmsl polypeptide comprises a mutation in a position corresponding to positions 701 or 922 of SmCmsl (SEQ ID NO: 10) or to positions 848 and 1213 of SulfCmsl (SEQ ID NO: 11) when aligned for maximum identity.
• Plasmid 131632 containing repair donor cassette (SEQ ID NO: 13), was designed with approximately 1,000-base pair homology upstream and downstream of the targeted site within the OsCAOl gene.
• the repair donor cassette included the maize ubiquitin promoter (SEQ ID NO:9) operably linked to a hygromycin resistance gene (SEQ ID NO:7, encoding SEQ ID NO:8), which was flanked at its 3' end by the Cauliflower Mosaic Virus 35S polyA sequence (SEQ ID NO:4).
• Plasmid 131592 was designed similarly to plasmid 131632, but without any homology arms up- or down-stream of the hygromycin cassette.
• plasmid 131592 contains nucleotides 1,001- 4,302 from SEQ ID NO: 13, including the maize ubiquitin promoter (SEQ ID NO:9) operably linked to a hygromycin resistance gene (SEQ ID NO:7, encoding SEQ ID NO:8), flanked at its 3' end by the Cauliflower Mosaic Virus 35S polyA sequence (SEQ ID NO:4).
• Unk2Cmsl (SEQ ID NO: 111, encoding SEQ ID NO: 111, encoding SEQ ID NO: 111
• Unkl6Cmsl (SEQ ID NO: 124, encoding SEQ ID NO: 124, encoding SEQ ID NO: 124
• Unk26Cmsl (SEQ ID NO: 134, encoding SEQ ID NO: 134, encoding SEQ ID NO: 134
• Unk33Cmsl (SEQ ID NO: 141, encoding SEQ ID NO: 141, encoding SEQ ID NO: 141
• LAHSCmsl (SEQ ID NO:18, encoding SEQ ID NO:22) 188 -431 (SEQ ID NO:29)
• Unk9Cmsl (SEQ ID NO:118, encoding SEQ ID NO:38) 222 -244 (SEQ ID NO:208)
• Unkl4Cmsl (SEQ ID NO:122, encoding SEQ ID NO:42) 233 -293 (SEQ ID NO:207)
• Unkl6Cmsl (SEQ ID NO:124, encoding SEQ ID NO:44) 238 -8 (SEQ ID NO:322)
• Unkl9Cmsl (SEQ ID NO:127, encoding SEQ ID NO:47) 241 -397/+356 (SEQ ID NO:215)
• Unk25Cmsl (SEQ ID NO:133, encoding SEQ ID NO:53) 253 -304 (SEQ ID NO:219)
• Unk36Cmsl (SEQ ID NO:144, encoding SEQ ID NO:64) 278 -26 (SEQ ID NO:324)
• Unk37Cmsl (SEQ ID NO: 145, encoding SEQ ID NO:65) 279 -16 (SEQ ID NO:323)
• T7EI analyses were performed to detect the presence of small insertions and/or deletions at the CAOl locus. T7EI analyses were performed as described previously (Begemann et al. (2017) Sci Reports 7: 11606). For callus samples whose T7EI analyses were indicative of a potential insertion or deletion, DNA sequencing analyses were performed to detect the presence of insertions and/or deletions at the CAOl locus.
• CRISPR nucleases are often classified by type, with, e.g., Cas9 nucleases classified as Type
• Unk32Cmsl (SEQ ID NO: 60) YGIDRGD MFLENKK KSGDDLA
• Unk49Cmsl (SEQ ID NO:225) YGIDRGD INLENLH KNSDDVA
• Unk54Cmsl (SEQ ID NO:229) FGLDNGE IVKEGFD HSNDGIA
• Unk58Cmsl (SEQ ID NO:234) YGIDRGI IYLENLE INYDSIA
• Unk61Cmsl (SEQ ID NO:237) YWIDKWT ICYETLD KSWDDLA
• Unk65Cmsl (SEQ ID NO:241) YGIDTGI ITIEYLD DSNDKVA
• Unk72Cmsl (SEQ ID NO:248) YGIDRGQ INLENLT KNSDEVA
• Unk75Cmsl (SEQ ID NO:251) YWFDKWE FVFEDKT HSWDDLA
• Unk78Cmsl (SEQ ID NO:254) YGIDRGE IILEDIE DDPDKVA
• Unk79Cmsl (SEQ ID NO:41) YGLDRGK VAFENLD DNSDKVA
• Unk41Cmsl (SEQ ID NO: 69) YGIDRGI IVLENIA RSGDQSA
• C2c3_CEPS (SEQ ID NO: 1 68 ) LAIDLGE PVLESSV GHADENA
• ObC2cl (SEQ ID NO: 1 60 ) LGVDLGT WIENLS MQADLNA
• DbC2cl (SEQ ID NO: 1 64 ) LSVDLGH WIENLA IHADLNA
• DtC2cl (SEQ ID NO: 159 ) LSVDLGV ILFEDLA IHADINA

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