LAWIT SHAI (US)
PHILLIPS JOAN MARIE (US)
SHEN BO (US)
ZHANG JUN (US)
US8395023B2 | 2013-03-12 | |||
US20160017349A1 | 2016-01-21 |
We claim: 1. A plant cell comprising a targeted genetic modification in a genomic locus of a gene encoding a polypeptide of interest, wherein the targeted genetic modification introduces into the genomic locus an endogenous microRNA recognition sequence, whereby expression of an endogenous microRNA that hybridizes to the endogenous microRNA recognition sequence decreases expression of the polypeptide of interest. 2. The plant cell of claim 1, wherein the microRNA recognition sequence is inserted into the 3’ untranslated region of the gene encoding the polypeptide of interest. 3. The plant cell of claim 1, wherein the microRNA recognition sequence is inserted into the 5’ untranslated region of the gene encoding the polypeptide of interest. 4. The plant cell of claim 1, wherein the microRNA recognition sequence is inserted into the coding region of the gene encoding the polypeptide of interest. 5. The plant cell of any one of claims 1-4, wherein the endogenous miRNA that hybridizes to the endogenous miRNA recognition sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 1-554. 6. The plant cell of any one of claims 1-5, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 7. The plant cell of claim 6 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 8. A plant comprising the plant cell of any one of claims 1-7. 9. A plant cell comprising a targeted genetic modification in the nucleotide sequence of an endogenous microRNA sequence, wherein the targeted genetic modification modifies the endogenous microRNA sequence to encode a modified microRNA that targets a genomic locus of a gene encoding a polypeptide of interest, whereby expression of the modified microRNA decreases expression of the polypeptide of interest. 10. The plant cell of claim 9, wherein the modified microRNA targets a sequence in the 3 untranslated region of the gene encoding the polypeptide of interest. 11. The plant cell of claim 9, wherein the modified microRNA targets a sequence in the 5’ untranslated region of the encoding the polypeptide of interest. 12. The plant cell of claim 9, wherein the modified microRNA targets a sequence in the coding region of the gene encoding the polypeptide of interest. 13. The plant cell of any one of claims 9-12, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 14. The plant cell of claim 13 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 15. The plant cell of any one of claims 9-14, wherein the endogenous miRNA sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 1-554. 16. A plant comprising the plant cell of any one of claims 9-15. 17. A seed produced by the plant of claim 8 or 16, wherein the seed comprises the targeted genetic modification. 18. A method of altering expression of a polypeptide of interest in a plant cell, the method comprising introducing in the plant cell a targeted genetic modification in a genomic locus of a gene encoding the polypeptide of interest, wherein the targeted genetic modification modifies the endogenous gene of interest to encode an endogenous microRNA recognition sequence. 19. The method of claim 18, wherein the microRNA recognition sequence is inserted into the 3’ untranslated region of the gene encoding the polypeptide of interest. 20. The method of claim 18, wherein the microRNA recognition sequence is inserted into the 5’ untranslated region of the gene encoding the polypeptide of interest. 21. The method of claim 18, wherein the microRNA recognition sequence is inserted into the coding region of the gene encoding the polypeptide of interest. 22. The method of any one of claims 18-21, wherein the endogenous miRNA recognition sequence comprises a nucleotide sequence that hybridizes to a nucleotide sequence of any one of SEQ ID NOs: 1-554. 23. The method of any one of claims 18-22, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 24. The method of claim 23 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 25. The method of any one of claims 18-24, wherein the targeted genetic modification is introduced using a genome modification technique selected from the group comprising a polynucleotide-guided endonuclease, CRISPR-Cas endonuclease, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site- specific meganucleases, or Argonaute. 26. A method of producing a plant having decreased expression of a polypeptide of interest, the method comprising: (a) introducing in a regenerable plant cell a targeted genetic modification at a genomic locus of a gene encoding the polypeptide of interest, wherein the targeted genetic modification modifies the genomic locus to encode an endogenous microRNA recognition sequence; and (b) generating the plant, wherein the plant comprises the targeted genetic modification. 27. The method of claim 26, wherein the microRNA recognition sequence is inserted into the 3’ untranslated region of the gene encoding the polypeptide of interest. 28. The method of claim 26, wherein the microRNA recognition sequence is inserted into the 5’ untranslated region of the gene encoding the polypeptide of interest. 29. The method of claim 26, wherein the microRNA recognition sequence is inserted into the coding region of the gene encoding the polypeptide of interest. 30. The method of any one of claims 26-29, wherein the endogenous miRNA recognition sequence comprises a nucleotide sequence that hybridizes to the nucleotide sequence of any one of SEQ ID NOs: 1-554. 31. The method of any one of claims 26-30, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 32. The method of claim 31 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 33. The method of any one of claims 26-32, wherein the targeted genetic modification is introduced using a genome modification technique selected from the group comprising a polynucleotide-guided endonuclease, CRISPR-Cas endonuclease, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site- specific meganucleases, or Argonaute. 34. A method of altering expression of a polypeptide of interest in a plant cell, the method comprising introducing in the plant cell a targeted genetic modification of an endogenous microRNA to produce a modified microRNA, wherein the modified microRNA targets a gene encoding the polypeptide of interest thereby reducing the expression of the polypeptide of interest. 35. The method of claim 34, wherein the modified microRNA targets a sequence in the 3’ untranslated region of the gene encoding the polypeptide of interest. 36. The method of claim 34, wherein the modified microRNA targets a sequence in the 5’ untranslated region of the gene encoding the polypeptide of interest. 37. The method of claim 34, wherein the modified microRNA targets a sequence in the coding region of the gene encoding the polypeptide of interest. 38. The method of any one of claims 34-37, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 39. The method of claim 38 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 40. The method of any one of claims 34-39, wherein the endogenous miRNA sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 1-554. 41. The method of any one of claims 34-40, wherein the targeted genetic modification is introduced using a genome modification technique selected from the group comprising a polynucleotide-guided endonuclease, CRISPR-Cas endonuclease, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site- specific meganucleases, or Argonaute. 42. A method of producing a plant having decreased expression of a polypeptide of interest, the method comprising: (a) introducing in a regenerable plant cell a targeted genetic modification in the nucleotide sequence of an endogenous microRNA, wherein the targeted genetic modification modifies the endogenous microRNA encode a modified microRNA that targets a gene encoding the polypeptide of interest; and (b) generating the plant, wherein the plant comprises the targeted genetic modification. 43. The method of claim 42, wherein the modified microRNA targets a sequence in the 3’ untranslated region of the gene encoding the polypeptide of interest. 44. The method of claim 42, wherein the modified microRNA targets a sequence in the 5’ untranslated region of the gene encoding the polypeptide of interest. 45. The method of claim 42, wherein the modified microRNA targets a sequence in the coding region of the gene encoding the polypeptide of interest. 46. The method of any one of claims 42-45, wherein the gene encoding the polypeptide of interest encodes a zinc finger containing protein, a kinase, a heat shock protein, a channel protein, an agronomic trait enhancing protein, an insect resistance protein, a disease resistance protein, a herbicide resistance protein, or a protein involved in sterility. 47. The method of claim 46 wherein the gene encoding the polypeptide of interest comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 564. 48. The method of any one of claims 42-47, wherein the endogenous miRNA sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 1-554. 49. The method of any one of claims 42-48, wherein the targeted genetic modification is introduced using a genome modification technique selected from the group comprising a polynucleotide-guided endonuclease, CRISPR-Cas endonuclease, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site- specific meganucleases, or Argonaute. |
[0085] As used herein, the terms “guide polynueleotide/Cas endonuclease complex”, “guide polynucieotide/Cas endonuclease system”, “ guide polynucleotide/Cas complex”, “guide polynudeotide/Cas system”, “guided Cas system” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that are capable of forming a complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the four known CRlSPR systems (Horvath and Barrangou, 2010, Science 327:167-170) such as a type I, II, or III CRISPR system. A Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity but can still specifically bind to a DNA target sequence when compiexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference).
[0086] A guide polynucieotide/Cas endonuclease complex can cleave one or both strands of a DNA target sequence. A guide polynucleotide/Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprise a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Appl. Publ. No. 2014/0189896, which is incorporated herein by reference. [0087] Other Cas endonuclease systems have been described in PCT patent applications PCT/US16/32073, filed May 12, 2016 and PCT/US16/32028 filed May 12, 2016, both applications incorporated herein by reference. [0088] “Cas9” (formerly referred to as Cas5, Csn1, or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence. Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA. [0089] Any guided endonuclease can be used in the methods disclosed herein. Such endonucleases include, but are not limited to, Cas9 and Cpf1 endonucleases. Many endonucleases have been described to date that can recognize specific PAM sequences (see for example –Jinek et al. (2012) Science 337 p 816-821, PCT patent applications PCT/US16/32073, filed May 12, 2016 and PCT/US16/32028 filed May 12, 2016 and Zetsche B et al.2015. Cell 163, 1013) and cleave the target DNA at a specific position. It is understood that based on the methods and embodiments described herein utilizing a guided Cas system one can now tailor these methods such that they can utilize any guided endonuclease system. [0090] The guide polynucleotide can also be a single molecule (also referred to as single guide polynucleotide) comprising a crNucleotide sequence linked to a tracrNucleotide sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a Cas endonuclease recognition domain (CER domain), that interacts with a Cas endonuclease polypeptide. By “domain” it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and/or RNA-DNA-combination sequence. The VT domain and /or the CER domain of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA-combination sequence. The single guide polynucleotide being comprised of sequences from the crNucleotide and the tracrNucleotide may be referred to as “single guide RNA” (when composed of a contiguous stretch of RNA nucleotides) or “single guide DNA” (when composed of a contiguous stretch of DNA nucleotides) or “single guide RNA-DNA” (when composed of a combination of RNA and DNA nucleotides). The single guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide/Cas endonuclease complex (also referred to as a guide polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the target site. (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 2015- 0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference.) [0091] The term “variable targeting domain” or “VT domain” is used interchangeably herein and includes a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double strand DNA target site. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof. [0092] The terms “single guide RNA" and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. [0093] The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Cas endonuclease system”, “ guide RNA/Cas complex”, “guide RNA/Cas system”, “gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease” , “RGEN” are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease that are capable of forming a complex , wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide RNA/Cas endonuclease complex herein can comprise Cas protein(s) and suitable RNA component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170) such as a type I, II, or III CRISPR system. A guide RNA/Cas endonuclease complex can comprise a Type II Cas9 endonuclease and at least one RNA component (e.g., a crRNA and tracrRNA, or a gRNA). (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference). [0094] The guide polynucleotide of the methods and compositions described herein may be any polynucleotide sequence that targets the genomic loci of a plant cell comprising a polynucleotide that encodes an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In certain embodiments, the guide polynucleotide is a guide RNA. The guide polynucleotide may also be present in a recombinant DNA construct. [0095] The guide polynucleotide can be introduced into a cell transiently, as single stranded polynucleotide or a double stranded polynucleotide, using any method known in the art such as, but not limited to, particle bombardment, Agrobacterium transformation or topical applications. The guide polynucleotide can also be introduced indirectly into a cell by introducing a recombinant DNA molecule (via methods such as, but not limited to, particle bombardment or Agrobacterium transformation) comprising a heterologous nucleic acid fragment encoding a guide polynucleotide, operably linked to a specific promoter that is capable of transcribing the guide RNA in said cell. The specific promoter can be, but is not limited to, a RNA polymerase III promoter, which allow for transcription of RNA with precisely defined, unmodified, 5’- and 3’- ends (DiCarlo et al., Nucleic Acids Res.41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161) as described in WO2016025131, published on February 18, 2016, incorporated herein in its entirety by reference. [0096] The terms “target site”, “target sequence”, “target site sequence, ”target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target locus” and “protospacer”, are used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, or any other DNA molecule in the genome (including chromosomal, choloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave . The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms endogenous target sequence” and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein. An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell. [0097] An “altered target site”, “altered target sequence”, “modified target site”, “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) – (iii). [0098] Methods for “modifying a target site” and “altering a target site” are used interchangeably herein and refer to methods for producing an altered target site. [0099] The length of the target DNA sequence (target site) can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The nick/cleavage site can be within the target sequence or the nick/cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other Cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5' overhangs, or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity and hence are capable of being recognized and cleaved by an Cas endonuclease. Assays to measure the single or double-strand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates containing recognition sites. [0100] A “protospacer adjacent motif” (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide/Cas endonuclease system described herein. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long. [0101] The terms “targeting”, “gene targeting” and “DNA targeting” are used interchangeably herein. DNA targeting herein may be the specific introduction of a knock-out, edit, or knock-in at a particular DNA sequence, such as in a chromosome or plasmid of a cell. In general, DNA targeting can be performed herein by cleaving one or both strands at a specific DNA sequence in a cell with an endonuclease associated with a suitable polynucleotide component. Such DNA cleavage, if a double-strand break (DSB), can prompt NHEJ or HDR processes which can lead to modifications at the target site. [0102] A targeting method herein can be performed in such a way that two or more DNA target sites are targeted in the method, for example. Such a method can optionally be characterized as a multiplex method. Two, three, four, five, six, seven, eight, nine, ten, or more target sites can be targeted at the same time in certain embodiments. A multiplex method is typically performed by a targeting method herein in which multiple different RNA components are provided, each designed to guide an guidepolynucleotide/Cas endonuclease complex to a unique DNA target site. [0103] The guide polynucleotide/Cas endonuclease system can be used in combination with a co-delivered polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest. (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and WO2015/026886 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference.) [0104] Various methods and compositions can be employed to obtain a cell or organism having a polynucleotide of interest inserted in a target site for a Cas endonuclease. Such methods can employ homologous recombination to provide integration of the polynucleotide of Interest at the target site. In one method provided, a polynucleotide of interest is provided to the organism cell in a donor DNA construct. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of Interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of Interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. By “homology” is meant DNA sequences that are similar. For example, a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome. A region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site. For example, the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5- 50, 5-55, 5-60, 5-65, 5- 70, 5-75, 5-80, 5- 85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5- 2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region. “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. The structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by the percent sequence identity over the whole length of the sequences, and/or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences. [0105] The amount of sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5–3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about 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%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Acid Probes, (Elsevier, New York). [0106] The structural similarity between a given genomic region and the corresponding region of homology found on the donor DNA can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of homology or sequence identity shared by the “region of homology” of the donor DNA and the “genomic region” of the organism 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, such that the sequences undergo homologous recombination [0107] The region of homology on the donor DNA can have homology to any sequence flanking the target site. While in some embodiments the regions of homology share significant sequence homology to the genomic sequence immediately flanking the target site, it is recognized that the regions of homology can be designed to have sufficient homology to regions that may be further 5' or 3' to the target site. In still other embodiments, the regions of homology can also have homology with a fragment of the target site along with downstream genomic regions. In one embodiment, the first region of homology further comprises a first fragment of the target site and the second region of homology comprises a second fragment of the target site, wherein the first and second fragments are dissimilar. [0108] As used herein, “homologous recombination” includes the exchange of DNA fragments between two DNA molecules at the sites of homology. [0109] Further uses for guide RNA/Cas endonuclease systems have been described (See U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015, WO2015/026886 A1, published on February 26, 2015, US 2015-0059010 A1, published on February 26, 2015, US application 62/023246, filed on July 07, 2014, and US application 62/036,652, filed on August 13, 2014, all of which are incorporated by reference herein) and include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and/or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest. [0110] Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published, among others, for cotton (U.S. Patent No. 5,004,863, U.S. Patent No.5,159,135); soybean (U.S. Patent No.5,569,834, U.S. Patent No. 5,416,011); Brassica (U.S. Patent No.5,463,174); peanut (Cheng et al., Plant Cell Rep.15:653 657 (1996), McKently et al., Plant Cell Rep.14:699703 (1995)); papaya (Ling et al., Bio/technology 9:752758 (1991)); and pea (Grant et al., Plant Cell Rep.15:254258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C.A., Mol. Biotechnol.16:5365 (2000). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F., Microbiol. Sci.4:2428 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira et al., Mol. Biotechnol.3:1723 (1995); Christou et al., Proc. Natl. Acad. Sci. U.S.A.84:39623966 (1987)), microinjection, or particle bombardment (McCabe et al., Biotechnology 6:923-926 (1988); Christou et al., Plant Physiol. 87:671674 (1988)). [0111] There are a variety of methods for the regeneration of plants from plant tissues. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, Eds.; In Methods for Plant Molecular Biology; Academic Press, Inc.: San Diego, CA, 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development or through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self- pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present disclosure containing a desired polypeptide is cultivated using methods well known to one skilled in the art. [0112] Terms used in the claims and specification are defined as set forth below unless otherwise specified. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety, for all purposes, to the same extent as if each were individually and specifically incorporated by reference. [0113] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLE 1 [0114] This example demonstrates the introduction of an endogenous microRNA recognition sequence to decrease expression of a gene of interest. [0115] The phytoene desaturase (PDS) gene encodes an essential plant carotenoid biosynthetic enzyme converting 15-cis-phytoene into zeta-carotene. PDS silenced plants display a photobleaching phenotype in leaves. To test whether the down-regulating expression of PDS through microRNA targeting can be achieved through placement of microRNA target site(s) within PDS’s expressed transcript the miR156B target site was introduced into the 3’UTR of the PDS gene. [0116] Gene editing via CRISPR-Cas9 was utilized to place the miR156B target site (SEQ ID NO: 555) into the 3’ untranslated region (3’UTR) of the Zea mays PDS gene (SEQ ID NO: 556) in a maize inbred. Guide RNA ZM-PDS-CR2 (SEQ ID NO: 557) created the double-strand break within the maize genome and homology-directed repair (HDR) using a 200-bp oligonucleotide template (SEQ ID NO: 558) inserted the miR156B target site into the maize PDS 3’UTR. The desired gene edit was confirmed by next generation sequencing of samples. [0117] Five tissue cultures samples showed strong chlorosis of early leaf tissue and all were found to have HDR edits containing the miR156B target site on both DNA strands, although not all edits had a perfect HDR matching the template. Fig.1 provides a representative example showing chlorosis of early leaf tissue in the bi-allelic HDR plants compared to the control non- edited plants. These HDR edited samples rapidly died as anticipated without functional levels of PDS. However, other edited plant seedlings advanced from tissue culture to the greenhouse. [0118] Further sequencing analysis of the edited seedlings advancing to the greenhouse showed that seven plants had one HDR allele with the inserted miR156B target site and one plant had both alleles edited; however, this seedling died after a few days in the greenhouse as expected. It is believed that the plants early survival was due to an unusually low level of miR156 expression in early tissue culture and vegetative phase allowing for some growth before chlorosis occurred. The other seven identified HDR edited plants had either a wildtype (WT) allele or a second edit involving simple SNPs. All still had one functioning PDS allele without miR156 regulation, allowing for normal plant growth and survival. [0119] Other locations within the PDS transcript were available for gene editing insertion of the miR156B target site, including within the 5’ untranslated region (5’UTR), the coding sequence, and other locations within the 3’UTR. All would be expected to have reduced PDS expression through regulation by miR156. Furthermore, regulation of PDS by other miRNAs such miR172 was considered. miR172 has a complementary expression pattern as compared with miR156. Its expression is highest in mature tissues and lowest in early vegetative tissue. Insertion of the miR172 target site into the PDS transcript would be expected to result in normal growth until adult phase, at which time chlorosis of tissue would be expected. [0120] Taken together, these results demonstrate that the introduction of an endogenous miRNA recognition sequence in a gene of interest results in decreased expression of the gene. EXAMPLE 2 [0121] The maize tasselless 1 (ZM-TSL1) gene when down-regulated reduces the size and appearance of a maize tassel. Down-regulation of the gene in multiple tissues throughout the plant’s growth cycle has negative pleiotropic effects on plant development. Therefore, we tested whether introducing a tassel preferred microRNA recognition sequence in the ZM-TSL1 gene would reduce the tassel size while eliminating other negative effects. [0122] Gene editing via CRISPR-Cas9 was utilized to place the tassel-specific miR529 target site (SEQ ID NO: 559) into the 3’ untranslated region (3’UTR) of the Zea mays TSL1 (SEQ ID NO: 560) in a maize inbred. Guide RNA ZM-TSL1-CR8 (SEQ ID NO: 561) created the double- strand break within the maize genome and homology-directed repair (HDR) using a 200-bp oligonucleotide template (SEQ ID NO: 563) inserted the miR529 target site into the maize TSL1 3’UTR. The template was designed to create as few alterations as possible when compared to the endogenous ZM-TSL1 sequence while allowing for the presence of the 21 bp miR529 target site within the 3’UTR. The design also altered one base in the PAM motif within the template in order to prevent further double stranded breaks within any edited plants. The desired gene edit was confirmed in twenty T0 seedlings by next generation sequencing of samples. Fifteen of those samples set seed, with resulting progeny still to be analyzed and phenotyped. [0123] Other locations within the ZM-TSL1 transcript were available for insertion of the miR529 target site by gene editing, including within the 5’ untranslated region (5’UTR), the coding sequence, and other locations within the 3’UTR. For example, guide RNA ZM-TLS1-CR9 (SEQ ID NO: 562) is also available within the 3’UTR providing a guide RNA site for miR529 target site insertion. Any miR529 target site insertions within the expressed ZM-TSL1 gene regardless of location would be expected to reduce TSL1 expression in the tassel without affecting ear growth. EXAMPLE 3 [0124] The maize NAC7 (ZM-NAC7) gene is a novel QTL controlling functional stay-green that was discovered in a mapping population derived from the Illinois High Protein 1 (IHP1) and Illinois Low Protein 1 (ILP1) lines, which show very different rates of leaf senescence. Transgenic maize lines where ZM-NAC7 was down-regulated by RNAi showed delayed senescence and increased both biomass and nitrogen accumulation in vegetative tissues, demonstrating that NAC7 functions as a negative regulator of the stay-green trait (J Zhang, et al, Plant Biotechnol J.201917(12):2272-2285). This example demonstrates utilizing the miR156e recognition sequence to regulate expression of endogenous ZM-NAC7. [0125] During early development in Arabidopsis, expression of miR156 is initially high and then steadily decreases as the plant matures (G Wu, et al, Cell, 2009, 138 (4): p750-759). Therefore, the insertion of the miRNA156 recognition sequence into the 3’ UTR of ZM-NAC7 should reduce the expression of ZM-NAC7 in the vegetative stage and increase photosynthesis, while maintaining certain endogenous ZM-NAC7 expression in the late developmental stage of maize to accelerate senescence and dry down grains. [0126] To insert the miRNA156e recognition sequence into ZM-NAC7, a guide RNA (SEQ ID NO: 566) was designed to target a sequence in the 3’-UTR of the ZM-NAC7 gene (SEQ ID NO: 565) in a maize inbred. The single guide RNA will create the double-strand break in ZM-NAC7 genomic DNA. Homology-directed repair using an oligonucleotide template containing the miR156e recognition sequence will insert the target site for miR156e (SEQ ID NO: 63). The desired gene edit will be confirmed by next generation sequencing of samples. Positive samples will be analyzed and phenotyped.
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