Genome editing to guide RNA capture

Abstract

The present disclosure provides methods and compositions for driving expression of coding sequences that have been integrated into the genome of a cell. The donor polynucleotide is integrated into the genome such that the non-functional promoter is located downstream of the genomic target sequence. The genomic target sequence can be bound by a site-specific promoter activation tool, wherein the site-specific promoter activation tool drives robust expression of a coding sequence operably linked to a non-functional promoter.

Description

Technical Field

[0001] The present disclosure relates generally to the field of plant molecular biology, and in particular to targeted modification of polynucleotides in plants.

[0002] Incorporated by Reference

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 369,550, filed on July 27, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0004] References to sequence listings

[0005] An official copy of the sequence listing is electronically submitted via EFS-Web as an XML-formatted sequence listing, with a file name of "9260-US-PSP.xml", created on July 17, 2023, and with a size of 100Kb, and is submitted simultaneously with this specification. The sequence listing contained in the XML-formatted file is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] The standard method of using site-specific nuclease to modify the genome of cells utilizes traditional methods that may limit speed and efficiency. These methods have a negative impact on the product development timeline. At present, site-specific nucleases are engineered to bind and cut genomic DNA, resulting in double-strand breaks in genomic DNA. The cellular mechanism repairs the double-strand breaks produced, and in the presence of donor polynucleotides, the donor polynucleotides can be integrated into the genomic DNA at the site of the double-strand breaks. These methods are inefficient and require screening of a large number of events to identify and separate events of genome insertion containing donor polynucleotides at specific sites in the genome of the cell. Therefore, there is still a need to develop a faster and more effective system for quickly and effectively inserting polynucleotide donors into specific sites in the genome of the cell. Summary of the invention

[0007] In some aspects, methods and compositions are provided.

[0008] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0009] The disclosure may be more fully understood from the following detailed description and accompanying drawings and sequence listing, which form a part of this application. These sequence descriptions and the accompanying sequence listing comply with the rules governing disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 C.FR §§1.821 and 1.825. These sequence descriptions contain the three-letter codes for amino acids as defined in 37 C.FR §§1.821 and 1.825, which are incorporated herein by reference.

[0010] Figure 1 A schematic diagram illustrating gene expression cassettes for use with the present disclosure is provided. Figure 1 Contains the following genetic elements and associated letter labels:

[0011] A: Zea mays ubiquitin promoter (SEQ ID NO: 9);

[0012] B: Zea mays ubiquitin 5'UTR (SEQ ID NO: 10);

[0013] C: Zea mays ubiquitin intron 1 (SEQ ID NO: 11);

[0014] D: maize optimized sequence encoding SV40 NLS (SEQ ID NO: 12);

[0015] E: Maize optimized gene (SEQ ID NO: 13) including ST-LS1 intron 2 encoding engineered variants of SpaCas12f1 (SEQ ID NO: 14-15);

[0016] F: maize optimized sequence encoding a dichotomous NLS (SEQ ID NO: 16);

[0017] G: maize optimized sequence encoding the CBF1 promoter activation domain (SEQ ID NO: 17);

[0018] H: maize ubiquitin terminator (SEQ ID NO: 18); and,

[0019] I: Zea mays optimized spycas9 gene including ST-LS1 intron 2 (SEQ ID NO: 19).

[0020] Figure 2 A schematic diagram illustrating gene expression cassettes for use with the present disclosure is provided. Figure 2 Contains the following genetic elements and associated letter labels:

[0021] A: Zea mays U6 promoter (including 3'G that promotes transcription) (SEQ ID NO: 20);

[0022] B: DNA sequences encoding SpyCas9 guide RNA variable targeting domains for target cleavage (SEQ ID NO: 21) or promoter activation (SEQ ID NO: 22-31);

[0023] C: DNA sequence encoding SpyCas9 gRNA minus the variable targeting domain (SEQ ID NO: 32);

[0024] D: Zea mays U6 terminator (SEQ ID NO: 33);

[0025] E: DNA sequence encoding SpaCas12f1 (SEQ ID NO: 34) or AsuCas12f1 (SEQ ID NO: 35) gRNA minus the variable targeting domain;

[0026] F: DNA sequences encoding SpaCas12f1 or AsuCas12f1 guide RNA variable targeting domains for target cleavage (SEQ ID NO: 36) or promoter activation (SEQ ID NO: 37-45); and,

[0027] G: DNA encoding ribozyme (SEQ ID NO: 46).

[0028] Figure 3 Schematic diagrams illustrating additional gene expression cassettes for use with the present disclosure are provided. Figure 3 Contains the following genetic elements and associated letter labels:

[0029] A: Zea mays ubiquitin promoter (SEQ ID NO: 9);

[0030] B: Zea mays ubiquitin 5'UTR (SEQ ID NO: 10);

[0031] C: Zea mays ubiquitin intron 1 (SEQ ID NO: 11);

[0032] D: Maize optimized spacas12f1 gene including ST-LS1 intron 2 (SEQ ID NOs: 14-15);

[0033] E: maize optimized sequence encoding SV40 NLS (SEQ ID NO: 12);

[0034] F: maize ubiquitin terminator (SEQ ID NO: 18);

[0035] G: Maize optimized gene including ST-LS1 intron 2 encoding nuclease inactive or dead (d) engineered variant of AsuCas12f1 (SEQ ID NO: 47);

[0036] H: maize optimized sequence encoding a bipartite NLS (SEQ ID NO: 16);

[0037] I: a maize optimized sequence encoding the CBF1 promoter activation domain (SEQ ID NO: 17); and,

[0038] J: Zea mays optimized nuclease dead (d) spycas9 gene including ST-LS1 intron 2 (SEQ ID NO: 48).

[0039] Figure 4 A schematic diagram illustrating a DNA repair template for use with the present disclosure is provided. Figure 4 Contains the following genetic elements and associated letter labels:

[0040] A: SpaCas12f1 or SpyCas9 DNA target cleavage site (SEQ ID NO: 50);

[0041] B: minimal 35S cauliflower mosaic virus promoter (SEQ ID NO: 51);

[0042] C: tobacco mosaic virus 5'UTR (SEQ ID NO: 52);

[0043] D: sequence encoding the DsRed visual selectable marker (SEQ ID NO: 53);

[0044] E: T28 terminator (SEQ ID NO: 54);

[0045] F: trait gene expression cassette (CXE20) (SEQ ID NO: 55);

[0046] G: 378 bp maize sequence (SEQ ID NO: 56) flanking the 5′ side of the genomic SpyCas9-gRNA DNA target cleavage site;

[0047] H: 419 bp maize sequence flanking the 3′ side of the genomic SpyCas9-gRNA DNA target cleavage site (SEQ ID NO: 57);

[0048] I: 70 bp maize sequence (SEQ ID NO: 66) flanking the 5' side of the genomic SpyCas9-gRNA DNA target cleavage site; and,

[0049] J: 175 bp maize sequence (SEQ ID NO: 67) flanking the 5' side of the genomic SpaCas12f1-gRNA DNA target cleavage site.

[0050] Figure 5 A schematic diagram illustrating the insertion of a donor polynucleotide from the T strand into the genomic DNA of a cell and a PCR assay for detecting the insertion is provided. Figure 5 Contains the following genetic elements and associated letter labels:

[0051] A: PCR amplicon of unique junction 1 generated by site-specific DNA insertion;

[0052] B: PCR amplicon of unique junction 2 generated by site-specific DNA insertion;

[0053] C: long PCR spanning the entire DNA insert; and,

[0054] Primers are represented as arrows and qPCR probes are represented as short dashed lines.

[0055] Figure 6 Provided is a schematic diagram illustrating the insertion of a donor polynucleotide from a T strand at a genome editing tool DNA cleavage site of a cell and the use of one or more nuclease-inactivated genome editing tools that can activate a promoter to select the insertion. The symbol of the line through the scissors shows the proximity of the nuclease-inactivated gene editing tool to a non-functional promoter. The nuclease-inactivated genome editing tool is used to activate a non-functional promoter to drive the robust expression level of a selectable marker, and is therefore referred to as a "promoter-activated genome editing tool" (site-specific promoter activation complex). Figure 6 Contains the following genetic elements and associated letter labels:

[0056] A: Cas-gRNA target cleavage site;

[0057] B: minimal 35S cauliflower mosaic virus promoter;

[0058] C: tobacco mosaic virus 5'UTR;

[0059] D: selectable marker;

[0060] E: T28 terminator;

[0061] F: desired trait gene expression cassette (SEQ ID NO: 55);

[0062] G: 419 bp maize sequence flanking the 3′ side of the genomic SpyCas9-Grna DNA target cleavage site (SEQ ID NO: 57);

[0063] H: 175 bp maize sequence (SEQ ID NO: 67) flanking the 5' side of the genomic SpaCas12f1-gRNA DNA target cleavage site; and,

[0064] The scissors symbol represents a Cas-gRNA complex capable of cleavage; and,

[0065] The symbol of a line passing through the scissors represents a promoter-activating Cas-gRNA complex (site-specific promoter activation complex).

[0066] Figure 7 It is shown how the directionality of insertion of a donor polynucleotide into the genomic DNA of a cell can be regulated by placing (relative to the positioning of the DNA cleavage site) one or more nuclease-inactivating genome editing tools capable of activating a promoter. Figure 7 Contains the following genetic elements and associated letter labels:

[0067] B: minimal 35S cauliflower mosaic virus promoter;

[0068] C: tobacco mosaic virus 5'UTR;

[0069] D: selectable marker;

[0070] E: T28 terminator;

[0071] F: desired trait gene expression cassette (SEQ ID NO: 55); and,

[0072] The symbol of a line passing through the scissors represents a promoter-activating Cas-gRNA complex (site-specific promoter activation complex).

[0073] Figure 8 Scarless excision of a selectable marker from genomic DNA is depicted. Figure 8 Contains the following genetic elements and associated letter labels:

[0074] B: minimal 35S cauliflower mosaic virus promoter;

[0075] C: tobacco mosaic virus 5'UTR;

[0076] D: selectable marker;

[0077] E: T28 terminator;

[0078] F: desired trait gene expression cassette (SEQ ID NO: 55);

[0079] G: Additional SpaCas12f1-gRNA target cleavage site for scarless excision of the selectable marker expression cassette (SEQ ID NO: 58);

[0080] H: maize sequence flanking the 5' side of the genomic SpaCas12f1-gRNA DNA target cleavage site (SEQ ID NO: 59); and,

[0081] The scissors symbol indicates the Cas-gRNA complex capable of cleavage.

[0082] Fig. 9The frequency of plants recovered containing targeted insertions is shown, as detected using PCR at the unique junctions ("junction 1" and "junction 2") created by DNA insertion cleavage of the DNA target site. The classical method does not use promoter activation of the selectable marker ("no promoter activation"). The method described herein utilizes "promoter activation".

[0083] Fig.10 An aspect of the method is provided, wherein first a gene expression cassette containing a non-functional promoter operably connected to a selectable marker (SM) is inserted into the border of a chromosome region predetermined for crossing. The 5' region of a non-functional promoter (MSNP) is targeted for Cas-gRNA cutting (as shown by a scissors symbol). The double-strand break produced causes the crossing of non-sister chromatids from the first homologous chromosome to the second homologous chromosome. After the non-sister chromatid crosses, the non-functional promoter operably connected to a selectable marker (SM) is located downstream of the genomic sequence that can be activated by a promoter to bind to the Cas-gRNA complex. Therefore, the crossover event causes the robust expression of the selectable marker, so that the cell containing this DNA repair result can be selected. Fig.10 Contains the following genetic elements and associated letter labels:

[0084] A: 5'Cas-gRNA target cleavage site of non-functional promoter;

[0085] B: selectable marker expression cassette with non-functional promoter;

[0086] The scissors symbol represents a Cas-gRNA complex capable of cleavage; and,

[0087] The symbol of a line passing through the scissors represents a promoter-activating Cas-gRNA complex (site-specific promoter activation complex).

[0088] Fig.11 Describe one aspect of the method, wherein first a gene expression cassette containing a non-functional promoter operably connected to a selectable marker (SM) is inserted into a chromosome so that it is reversed relative to the position of the genomic sequence that can be activated by the promoter Cas-gRNA complex. The double-strand breaks at both ends of the chromosome region to be inverted release it from the genomic DNA. DNA repair events cause the selectable marker cassette and the associated chromosomal DNA to be inverted, so that the non-functional promoter operably connected to the selectable marker (SM) is close to one or more promoters activating the Cas-gRNA complex. Therefore, the inversion event causes the robust expression of the selectable marker, so that cells with this DNA repair result can be selected. Fig.11 Contains the following genetic elements and associated letter labels:

[0089] A: 5'Cas-gRNA target cleavage site of non-functional promoter;

[0090] B: selectable marker expression cassette with non-functional promoter;

[0091] C: Cas-gRNA target cleavage site at the distal boundary of the target region;

[0092] The scissors symbol represents a Cas-gRNA complex capable of cleavage; and,

[0093] The symbol of a line passing through the scissors represents a promoter-activating Cas-gRNA complex (site-specific promoter activation complex).

[0094] Fig.12 One aspect of this method is shown, wherein a gene expression cassette containing a non-functional promoter operably linked to a selectable marker (SM) is first inserted into a first chromosome (e.g. Fig.12 The second chromosome (e.g. Fig.12 The region on chromosome 2) and the gene expression cassette of chromosome 1 are targeted for Cas-gRNA cleavage (as indicated by the scissors symbol). The resulting double-strand break releases the gene expression cassette from chromosome 1 so that it can be inserted into chromosome 2. After the gene expression cassette is inserted into chromosome 2, a non-functional promoter operably linked to a selectable marker (SM) is located downstream of the genomic sequence that can be activated by the promoter to bind the Cas-gRNA complex. Therefore, the crossover event results in robust expression of the selectable marker, so that cells containing this DNA repair result can be selected. Fig.12 Contains the following genetic elements and associated letter labels:

[0095] A: 5'Cas-gRNA target cleavage site of non-functional promoter;

[0096] B: selectable marker expression cassette with non-functional promoter;

[0097] C: Cas-gRNA target cleavage site at the distal boundary of the target region;

[0098] D: Cas-gRNA target cleavage site at a new chromosomal location;

[0099] The scissors symbol represents a Cas-gRNA complex capable of cleavage; and,

[0100] The symbol of a line passing through the scissors represents a promoter-activating Cas-gRNA complex (site-specific promoter activation complex). DETAILED DESCRIPTION

[0101] Provided are various compositions and methods for effectively inserting a polynucleotide donor at a specific site in the genome of a cell (e.g., a plant cell). On the one hand, a gene expression cassette is designed, which contains a non-functional promoter that drives the expression of a coding sequence. After the gene expression cassette is inserted into the desired genomic position, a site-specific promoter activation complex (non-limiting example; promoter activation Cas and gRNA complex) is combined with the region flanking the genomic insertion site, and is used to drive a non-functional promoter to robustly express the coding sequence. Various applications of the method can be used for different genomic configurations. For example, the method can utilize genome crossing, inversion, and repositioning to locate a non-functional promoter near the genomic region bound by the site-specific promoter activation complex. Once the site-specific promoter activation complex approaches the non-functional promoter, the expression of the selectable marker is driven at a robust level, thereby allowing selection based on the marker. These new methods and compositions provide donor-mediated genome insertion, improved efficiency of genome editing, improved percentage of regenerated transformed cells (e.g., plant cells), lower loss rate of target cells / organisms, removal of the cumbersome yield, and reduced unwanted DNA integration.

[0102] definition

[0103] Unless otherwise specified, the terms used in the claims and specification are defined as set forth below. It must be noted that, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0104] The term "genome" refers to the entire complementary sequence of genetic material (genes and non-coding sequences) present in each cell of an organism or virus or organelle; and / or a complete set of chromosomes inherited from one parent as a (haploid) unit. The term "genome" when applied to prokaryotic or eukaryotic cells or organismal cells not only encompasses the chromosomal DNA found in the nucleus, but also encompasses the organelle DNA found in the subcellular components of the cell (e.g., mitochondria, or plastids).

[0105] The terms "provided (to)" and "introduced (to)" are used interchangeably herein. In another aspect, it means that a particular composition is functionally associated with a cell or other molecule. In one aspect, it means that a particular composition is absorbed into the interior of a cell.

[0106] The term "endogenous" means a sequence or other molecule that is naturally present in a cell or organism. In one aspect, an endogenous polynucleotide is typically found in the genome of a cell; that is, not heterologous.

[0107] The term "heterologous" refers to the difference between the original environment, position, or composition of a particular polynucleotide or polypeptide sequence and its current environment, position, or composition. Non-limiting examples include taxonomic derivation (e.g., if a polynucleotide sequence obtained from maize is inserted into the genome of a different variety or cultivar of a rice (Oryza sativa) plant or maize, the polynucleotide sequence will be heterologous; or a polynucleotide obtained from a bacterium is introduced into a plant's cell) or a sequence (e.g., a polynucleotide sequence is obtained from maize, separated, modified, and reintroduced into a maize plant). As used herein, "heterologous" with respect to a sequence can refer to a sequence that is derived from a different species, variant, exotic species, or, if derived from the same species, is substantially modified from its native form in a composition and / or genomic locus by deliberate human intervention. For example, the promoter operably linked to the heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or, if from the same / similar species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide. Alternatively, one or more compositions, such as those provided herein, can be completely synthetic.

[0108] As used herein, "nucleic acid" means polynucleotides, and includes a single-stranded polymer or a double-stranded polymer of a deoxyribonucleotide base or a ribonucleotide base. Nucleic acids can also include fragments and modified nucleotides. Therefore, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" and "nucleic acid fragment" are used interchangeably to represent single-stranded or double-stranded RNA and / or DNA and / or RNA-DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0109] The relationship between two or more polynucleotides or polypeptides can be determined. The structural relationship between polynucleotides and polypeptide sequences, fragments thereof, variants thereof, and these sequences can be described by the terms "homology", "homologous", "substantially identical", "substantially similar", and "substantially corresponding", which are used interchangeably in this article. These refer to polypeptides or nucleic acid sequences in which changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or produce a certain phenotype. These terms also refer to one or more modifications of the nucleic acid sequence that do not substantially change the functional properties of the resulting nucleic acid relative to the initial unmodified nucleic acid. These modifications include deletions, substitutions, and / or insertions of one or more nucleotides in the nucleic acid fragment.

[0110] Sequence relationships can be defined by comparison of their composition, or by their ability to hybridize, or by their ability to participate in homologous recombination.

[0111] Alignment methods for sequences for comparison are well known in the art. Therefore, the determination of the percent sequence identity between any two sequences can be performed using a mathematical algorithm. Sequence alignment and percent identity or similarity calculations can be determined using a variety of comparison methods designed to detect homologous sequences, including but not limited to the MegAlign program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). In the context of this application, it should be understood that when using sequence analysis software to analyze, the result of the analysis will be based on the "default value" of the program of reference, unless otherwise stated. As used herein, "default value" will mean any set of values ​​or parameters initially loaded into the software when initialized for the first time.

[0112] In the context of nucleic acid or polypeptide sequence, "sequence identity" or "identity" refers to the nucleic acid bases or amino acid residues in two sequences that are identical when aligned for maximum correspondence on a specified comparison window. The term "percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences on a comparison window, wherein for the optimal comparison of two sequences, compared with a reference sequence (which does not include additions or deletions), the portion of a polynucleotide or polypeptide sequence in a comparison window may include additions or deletions (i.e., spaces). The percentage is calculated in the following manner: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and then these results are multiplied by 100 to produce the percentage of sequence identity. Useful examples of percentage sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50% to 100%. Any program described herein can be used to determine these identities.

[0113] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and found in the MegAlign program of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). For multiple alignments, the default values ​​correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. The default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5. For nucleic acids, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4, and STORED DIAGONAL=4. After aligning sequences using the Clustal V program, it is possible to obtain "percent identity" by viewing the "Sequence Distances" table in the same program. The "Clustal W method of alignment" corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and is found in the MegAlign v6.1 program of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). Default parameters for multiple alignment (Gap Penalty = 10, Gap Length Penalty = 0.2, Delay Divergen Seqs (%) = 30, DNA Transformation Weight = 0.5, Protein Weight Matrix = Gonnet Series, DNA Weight Matrix = IUB). After aligning the sequences using the Clustal W program, it is possible to obtain "Percent Identity" by viewing the "Sequence Distances" table in the same program.Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for nucleotide sequences using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a gap creation penalty weight of 8 and a gap length extension penalty weight of 2 and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 10915). GAP uses the algorithm of Needleman and Wunsch, (1970) J Mol Biol [Journal of Molecular Biology] 48: 443-53 to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible comparisons and gap positions, and uses the gap generation penalty and gap extension penalty in the unit of matching bases to produce the comparison with the maximum number of matching bases and the minimum gap. " BLAST " is the search algorithm for finding the region of similarity between biological sequences provided by the National Center for Biotechnology Information (NCBI) of the United States. The program compares nucleotide or protein sequence with the sequence database, and calculates the statistical significance of matching to identify the sequence with enough similarities to the query sequence, so that similarity will not be predicted as having occurred randomly. The sequence identified by BLAST report and their local comparison with the query sequence. As used herein, " sequence identity percentage " means the value determined by comparing two aligned sequences on a comparison window, wherein compared with the reference sequence (which does not include additions or deletions), the polynucleotide sequence portion in this comparison window can include additions or deletions (i.e., gaps), to perform the best comparison of these two sequences. The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then multiplying the result by 100 to produce the percentage of sequence identity.

[0114] It is well understood by those skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified natural or synthetic polypeptides that have the same or similar function or activity. Useful examples of percentage identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50% to 100%. In fact, any integer amino acid identity from 50% to 100% will be useful in describing the present disclosure, for example, 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%.

[0115] "Homologous" means that the DNA sequences are similar. For example, a "region homologous to a genomic region" found on a donor DNA is a region of DNA having a similar sequence to a given "genomic sequence" in the genome of a cell or organism. A homologous region can have any length sufficient to promote homologous recombination at the target site of cleavage. For example, the length of a homologous region can include 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, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-2000 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-210, 1-220, 1-230, 1-240, 1-250, 1-260, 1-270, 1-280, 1-290, 1-300, 1-310 or more bases, so that the homologous region has enough homology to carry out homologous recombination with the corresponding genomic region." enough homology" means that two polynucleotide sequences have enough structural similarities to serve as substrates for homologous recombination reactions. Structural similarity includes the total length of each polynucleotide fragment and the sequence similarity of the polynucleotides. Sequence similarity can be described by percent sequence identity over the entire length of the sequence and / or by conserved regions that include local similarity (eg, contiguous nucleotides with 100% sequence identity) as well as percent sequence identity over a portion of the length of the sequence.

[0116] As used herein, "isolated" polynucleotides or polypeptides or their biologically active parts are substantially or essentially free of components that normally accompany or interact with polynucleotides or polypeptides as found in their naturally occurring environment. Therefore, isolated or purified polynucleotides or polypeptides are substantially free of other cellular materials or culture medium components when produced by recombinant technology, or substantially free of chemical precursors or other molecules when chemically synthesized. Optimally, "isolated" polynucleotides do not contain sequences (that is, sequences at the 5' and 3' ends of the polynucleotides) that are naturally flanking the polynucleotides in the genomic DNA of the organism from which the polynucleotides are derived (optimally protein coding sequences). For example, in various embodiments, the isolated polynucleotides can include a nucleotide sequence less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb, and in the genomic DNA of the cell from which the polynucleotides are derived, the nucleotide sequence is naturally located at the flank of the polynucleotides. A polypeptide that is substantially free of cellular material includes preparations of the polypeptide having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When a polypeptide of the invention or a biologically active portion thereof is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target protein molecules.

[0117] As used herein, a polynucleotide or polypeptide is "recombinant" when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, the following polynucleotide is a recombinant polynucleotide that is inserted into a vector or any other heterologous location (e.g., in the genome of another organism) such that the polynucleotide is not associated with nucleotide sequences that flank the polynucleotide that are typically found in nature. A polypeptide expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Similarly, polynucleotide sequences that do not occur naturally, such as variants of naturally occurring genes, are recombinant.

[0118] The terms "recombinant polynucleotide", "recombinant nucleotide", "recombinant DNA" and "recombinant DNA construct" are used interchangeably herein. Recombinant constructs include nucleic acid sequences, such as artificial or heterologous combinations of regulatory sequences and coding sequences that are not found together in nature. For example, a transfer box can include a target restriction site and a heterologous polynucleotide. In other embodiments, the recombinant construct may include regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that of naturally occurring. Such constructs can be used alone or in combination with a vector. If a vector is used, the selection of the vector depends on the method that will be used to transform the host cell as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled person fully understands the genetic elements that must be present on the vector in order to successfully transform, select and propagate the host cell containing any isolated nucleic acid fragment provided herein. The skilled person will also recognize that different independent transformation events will result in different expression levels and patterns (Jones et al., EMBO J. [Journal of the European Molecular Biology Association] 4: 2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics [Molecular Genetics and General Genetics] 218: 78-86 (1989)), and therefore multiple events must be screened to obtain lines that show the desired expression levels and patterns. Such screening can be accomplished by DNA blot analysis, Northern analysis of mRNA expression, immunoblot analysis of protein expression, or phenotypic analysis, etc.

[0119] A "centimorgan" (cM) or "map distance unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, loci, or any pair thereof, at which 1% of the products of meiosis are recombinant. Thus, one centimorgan is equivalent to a distance equal to the 1% average recombination frequency between two linked genes, markers, target sites, loci, or any pair thereof.

[0120] "Open reading frame" is abbreviated as ORF.

[0121] "Gene" includes a nucleic acid fragment that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). "Native gene" refers to a gene found in its natural endogenous location with its own regulatory sequences.

[0122] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are identical, the plant is homozygous at that locus. If the alleles present at a given locus on a chromosome are different, the plant is heterozygous at that locus.

[0123] In the context of a pair of homologous chromosomes, the term "homologous" refers to a pair of chromosomes from an individual, whose length, gene position and centromere position are similar, and are arranged and synapsed during meiosis. In an individual, a chromosome in a pair of homologous chromosomes comes from the mother of the individual (that is, "maternally derived"), and another chromosome in the pair of chromosomes comes from the father (that is, "paternally derived"). In the context of genes, the term "homologous" refers to a pair of genes in which each gene is located at the same position in each homologous chromosome and has the same function. The term "homologous recombination" refers to the mutual exchange between homologous chromosomes (for example, between the non-sister chromatids of homologous chromosomes during meiosis) at the corresponding position. Homologous recombination can also occur in somatic cells during mitosis (somatic cell crossing).

[0124] "Coding sequence" refers to a polynucleotide sequence that encodes a specific amino acid sequence. "Regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internal nucleotide sequence, or downstream nucleotide sequence (3' non-coding sequence) of a coding sequence, and it affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5' non-translated sequences, 3' non-translated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0125] A "mutated gene" or a gene that has been "mutated" is a gene that has been altered by human intervention. Such a "mutated gene" has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion or substitution. In certain embodiments of the present disclosure, the mutated gene comprises an alteration caused by a guide polynucleotide / Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising a mutated gene.

[0126] As used herein, the term "morphogenetic gene" means a gene that, when ectopically expressed, stimulates the formation of a somatic cell-derived structure that can produce a plant. More precisely, the ectopic expression of a morphogenetic gene stimulates the de novo formation of a somatic embryo or organogenesis structure (e.g., bud meristem) that can produce a plant. This stimulated de novo formation occurs in the cell expressing the morphogenetic gene or in a neighboring cell. A morphogenetic gene can be a transcription factor that regulates the expression of other genes, or a gene that affects hormone levels in plant tissues, both of which can stimulate morphological changes. As used herein, the term "morphogenetic factor" means a morphogenetic gene and / or a protein expressed by a morphogenetic gene.

[0127] "Promoter" is a DNA region that participates in the recognition and combination of RNA polymerase and other proteins to initiate transcription. The promoter sequence is composed of a proximal upstream element and a distal upstream element, and the latter element is generally referred to as an enhancer. "Enhancer" is a DNA sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. Promoter can be derived from natural genes in its entirety, or be composed of different elements derived from different promoters existing in nature, and / or comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may guide gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. It is further recognized that, since the exact boundaries of regulatory sequences are not yet fully defined in most cases, some DNA fragments of variations may have the same promoter activity.

[0128] Promoters that cause gene expression in most cell types under most circumstances are generally referred to as "constitutive promoters". The term "inducible promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, such as by a chemical compound (chemical inducer), or in response to environmental, hormone, chemical, and / or developmental signals. Inducible or regulated promoters include promoters that are induced or regulated, for example, by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or chemicals such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners.

[0129] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the upstream of the translation initiation sequence in the mRNA. The translation leader sequence can affect the processing of the primary transcript to the mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol [molecular biotechnology] 3: 225 236).

[0130] As used herein, "minimal promoter" or substantially similar terms refer to promoter elements, particularly TATA elements, that are inactive or have reduced and lower levels of promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to allow transcription. Typically, a minimal promoter is a minimal continuous stretch of DNA sequence sufficient to direct the RNA polymerase II machinery to accurately initiate transcription (for review, see: Struhl, 1987, Cell 49: 295-297; Smale, 1994, Transcription: Mechanisms and Regulation (eds. RC Conaway and JW Conaway), pp. 63-81 / Raven Press, Ltd., New York; Smale, 1997, Biochim. Biophys. Acta 1351: 73-88; Smale et al., 1998, Cold Spring Harbor Laboratory Press, New York, NY). Harb.Symp.Quant.Biol. [Cold Spring Harbor Quantitative Biology Symposium] 58:21-31; Smale, 2001, Genes & Dev. [Genes and Development] 15:2503-2508; Weis and Reinberg, 1992, FASEB J. [Journal of the Federation of American Societies for Experimental Biology] 6:3300-3309; Burke et al., 1998, Cold Spring Harb.Symp.Quant.Biol [Cold Spring Harbor Quantitative Biology Symposium] 63:75-82). Several sequence motifs are common in minimal promoters, including the TATA box, the initiator (Inr), the TFIIB recognition element (BRE), and the downstream promoter element (DPE). However, not all of these elements will appear in all promoters. That is, there is no universal minimal promoter. In some cases, the minimal promoter can be referred to as a "core promoter".

[0131] "3' non-coding sequence", "transcription terminator" or "termination sequence" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is generally characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.

[0132] "RNA transcript" refers to the product produced by the transcription catalyzed by RNA polymerase of a DNA sequence. When the RNA transcript is a fully complementary copy of a DNA sequence, the RNA transcript is referred to as a primary transcript or pre-mRNA. When the RNA transcript is an RNA sequence derived from the post-transcriptional processing of the pre-mRNA of the primary transcript, the RNA transcript is referred to as a mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to an RNA that does not contain introns and can be translated into protein by a cell. "cDNA" refers to a DNA that is complementary to an mRNA template and synthesized from an mRNA template using a reverse transcriptase. The cDNA can be single-stranded or can be converted into a double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that includes mRNA and can be translated into protein in a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks the expression of a target gene (see, e.g., U.S. Patent No. 5,107,065). Antisense RNA can be complementary to any part of a particular gene transcript, i.e., 5' non-coding sequence, 3' non-coding sequence, intron or coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that can be untranslated but still have an effect on cellular processes. The terms "complementary sequence" and "reverse complementary sequence" are used interchangeably herein with respect to mRNA transcripts and mean antisense RNA that defines the messenger.

[0133] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. The 7-methylguanylate residue is located at the 5' end of messenger RNA (mRNA) in eukaryotes. In eukaryotes, RNA polymerase II (Pol II) transcribes mRNA. Messenger RNA capping typically occurs as follows: the very terminal 5' phosphate group of the mRNA transcript is removed with an RNA terminal phosphatase, leaving two terminal phosphates. Guanosine monophosphate (GMP) is added to the terminal phosphate of the transcript with a guanylyltransferase, leaving a 5'-5' triphosphate-linked guanine at the end of the transcript. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.

[0134] The term "without a 5'-cap" herein is used to refer to an RNA having, for example, a 5'-hydroxyl group instead of a 5'-cap. For example, such an RNA may be referred to as an "uncapped RNA". Because 5'-capped RNA has a tendency to be exported from the nucleus, uncapped RNA may accumulate better in the nucleus after transcription. One or more RNA components herein are uncapped.

[0135] The term "operably connected" refers to the association of a nucleic acid sequence on a single nucleic acid fragment so that the function of one of the nucleic acid sequences is regulated by another nucleic acid sequence. For example, when a promoter is capable of regulating the expression of a coding sequence (that is, the coding sequence is under the transcriptional control of a promoter), the promoter is operably connected to the coding sequence. The coding sequence can be operably connected to a regulatory sequence in a sense or antisense orientation. In another example, a complementary RNA region can be directly or indirectly operably connected to or within a target mRNA at 5' or 3' of a target mRNA, or the first complementary region is at 5' of the target mRNA and its complementary sequence is at 3' of the target mRNA.

[0136] As used herein, the term "expression" refers to the production of a functional end-product (eg, mRNA, guide RNA, or protein) in either precursor or mature form.

[0137] "Domain" means a continuous stretch of nucleotides (which may be RNA, DNA, and / or RNA-DNA combination sequences) or amino acids.

[0138] As used herein, "expression" at a "higher level" refers to enhancing transcription or translation by binding a site-specific promoter activation complex to a specific site on DNA or mRNA. Preferably, "expression" at a "higher level" includes a significant change of at least 1% increase in transcription or translation levels.

[0139] The term "conserved domain" or "motif" means a group of polynucleotides or amino acids that are conserved at specific positions along the aligned sequences of evolutionarily related proteins. Although amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, stability, or activity of the protein. Because they are identified by their high conservation in the aligned sequences of a family of protein homologs, they can be used as identifiers or "signatures" to determine whether a protein with a newly determined sequence belongs to a previously identified protein family.

[0140] The term "fragment" refers to a group of continuous polynucleotides or polypeptides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous polynucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous polypeptides. A fragment may or may not exhibit the function of a sequence having a certain percentage of identity over the length of the fragment.

[0141] The terms "functionally equivalent fragments", "functional fragments" and "functional equivalent fragments" are used interchangeably herein. These terms refer to a portion or subsequence of a nucleic acid fragment or polypeptide that exhibits the same activity or function as the longer sequence from which it is derived. In one example, whether or not the fragment encodes a complete protein found in nature, the fragment retains the ability to alter gene expression, produce double-stranded gaps or produce a certain phenotype. In some aspects, partial activity is retained. In some aspects, all activity is retained.

[0142] The terms "functionally equivalent variants", "functional variants" and "functional equivalent variants" are used interchangeably herein. These terms refer to nucleic acid fragments or polypeptides that have the same activity or function as the source sequence from which they are derived, but differ from the source sequence by at least one nucleotide or amino acid. In one example, the variants all retain the ability to alter gene expression, produce double-stranded gaps or produce a certain phenotype. In some aspects, partial activity is retained. In some aspects, all activity is retained.

[0143] A functional fragment or functional variant has at least 50%, between 50% and 55%, at least 55%, between 55% and 60%, at least 60% or more of the same sequence as a naturally derived polynucleotide or polypeptide of at least 50, 50 to 100, at least 100, 100 to 150, at least 150, 150 to 200, at least 200, 200 to 250, at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, 400 to 450, at least 50 ... , between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, 99% and 100%, or 100% sequence identity and retain at least partial activity.

[0144] "Modified," "edited," or "altered" with respect to a polynucleotide or target sequence refers to a nucleotide sequence that comprises at least one alteration when compared to its unmodified nucleotide sequence. Such "alterations" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) association of another molecule or atom via covalent, ionic or hydrogen bonding, or (v) any combination of (i)-(iv).

[0145] Proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of one or more proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance for amino acid substitutions that are unlikely to affect the biological activity of a protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC [National Biomedical Research Foundation, Washington, DC, USA]. Conservative substitutions, such as exchanging one amino acid for another with similar properties, would be preferred. Conservative deletions, insertions, and amino acid substitutions are expected to produce no radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by conventional screening assays. Assays for double-strand-break-inducing activity are known, and generally measure the overall activity and specificity of an agent for a DNA substrate comprising a target site.

[0146] A "mature" protein refers to a post-translationally processed polypeptide (ie, a polypeptide from which any pre-peptide or propeptide present in the primary translation product has been removed).

[0147] "Precursor" protein refers to the primary product of translation of mRNA (ie, pre- or propeptides are still present). Pre- or propeptides may be, but are not limited to, intracellular localization signals.

[0148] An "optimized" polynucleotide is a sequence that has been optimized for improved expression in a specific heterologous host cell.

[0149] A "codon-modified gene" or "codon-biased gene" or "codon-optimized gene" is a gene whose frequency of codon usage has been designed to mimic the frequency of preferred codon usage of the host cell.

[0150] A "plant optimized nucleotide sequence" is a nucleotide sequence optimized for expression in plants (particularly for increased expression in plants). Plant optimized nucleotide sequences include codon optimized genes. Plant optimized nucleotide sequences can be synthesized by modifying the nucleotide sequence encoding a protein (such as the Cas endonucleases disclosed herein) using one or more plant preferred codons to improve expression. For a discussion of the use of host preference, see, e.g., Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11.

[0151] The terms "plasmid", "vector" and "box" refer to linear or circular extrachromosomal elements, which usually carry genes that are not part of the central metabolism of the cell, and are usually in the form of double-stranded DNA. Such elements can be autonomous replication sequences, genome integration sequences, phages, or nucleotide sequences derived from any source, single-stranded or double-stranded DNA or RNA, in linear or circular form, many of which have been connected or recombined into unique structures that can introduce the polynucleotide of interest into cells." transformation box" refers to a specific vector that contains genes and has elements other than genes that promote transformation of specific host cells. "expression cassette" refers to a specific vector that contains genes and has elements other than genes that allow expression of the gene in a host.

[0152] "Purpose polynucleotide" includes any nucleotide sequence encoding a protein or polypeptide that improves the desirability of an organism, such as an animal or plant. Purpose polynucleotide: includes, but is not limited to, polynucleotides encoding important traits for agronomy, herbicide-resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic markers, or any other trait of important agronomic or commercial significance. Purpose polynucleotides may additionally be utilized in sense or antisense orientations. In addition, more than one purpose polynucleotide may be utilized together or "stacked" to provide additional benefits.

[0153] As used herein, a "target genomic region" is a segment of a chromosome in a plant genome that is desirable for introducing a double-strand break, a target polynucleotide, or a target trait. The target genomic region may include, for example, one or more target polynucleotides. Typically, the target genomic region of the present invention comprises a chromosome segment of 0-15 centimorgans (cM).

[0154] The terms "knockout," "gene knock-out," and genetic knock-out are used interchangeably herein. Knockout refers to a DNA sequence in a cell that has been rendered partially or completely ineffective by targeting with a DSB agent; for example, the DNA sequence may have encoded an amino acid sequence prior to knockout, or may have had a regulatory function (e.g., a promoter).

[0155] The terms "knock-in", "gene knock-in", "gene insertion" and "gene knock-in" are used interchangeably herein. Knock-in refers to the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a DSB agent (e.g., by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used). Examples of knock-ins are the specific insertion of a heterologous amino acid coding sequence in the coding region of a gene, or the specific insertion of a transcriptional regulatory element in a genetic locus.

[0156] "Introducing" is intended to mean providing a polynucleotide or polypeptide or polynucleotide-protein complex to a target, such as a cell or organism, in such a way that the component or components gain access to the interior of a cell of the organism or to the cell itself.

[0157] Typically, "host" refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell) in vivo or in vitro, or a cell (e.g., a cell line) from a multicellular organism cultured as a unicellular entity, into which a heterologous polynucleotide or polypeptide has been introduced. In some embodiments, the cell is selected from the group consisting of: a primitive cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algae cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, a cattle cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In some cases, the cell is an in vitro cell. In some cases, the cell is an in vivo cell.

[0158] As used herein, the terms "target site," "target sequence," and "target polynucleotide" are used interchangeably herein and refer to a polynucleotide sequence in the genome of a plant cell or yeast cell that contains a recognition site for a double-strand break inducing agent.

[0159] A "target cell" is a cell that contains a target sequence and is the subject of receiving a specific double-strand break-inducing agent.

[0160] A "break-inducing agent" is a composition that produces a cut in at least one strand of a polynucleotide. In some aspects, a break-inducing agent may be capable of, or have its activity altered such that it is capable of producing a break in only one strand of a polynucleotide. Producing a single-strand break in a double-stranded target sequence may be referred to herein as "nicking" the target sequence.

[0161] As used herein, the term "double-strand break inducing agent" or equivalently "double-strand break agent" or "DSB agent" refers to any composition that produces a double-strand break in a target polynucleotide sequence (i.e., a break in both chains of a double-stranded polynucleotide). Examples of DSB agents include, but are not limited to, meganucleases, TAL effector nucleases, Argonautes, zinc finger nucleases, and Cas endonucleases (alone or as part of a ribonucleoprotein complex). Producing a double-strand break in a target sequence may be referred to herein as "cutting" or "cutting" the target sequence. In some aspects, a DSB agent is a nuclease. In some aspects, a DSB agent is an endonuclease. "Endonuclease" refers to an enzyme that cuts a phosphodiester bond within a polynucleotide chain. In some embodiments, a double-strand break results in a "flat end" of a double-stranded polynucleotide, in which the two chains are sheared directly across each other without producing nucleotide overhangs. When a first cleavage of a first polynucleotide backbone occurs between a first set of two nucleotides on one strand, and a second cleavage of a second polynucleotide backbone occurs between a second set of two nucleotides on the opposite strand, a "blunt" end shearing of a double-stranded polynucleotide is produced, wherein each of the first set of two nucleotides hydrogen bonds with one of the second set of two nucleotides, resulting in a sheared strand having no nucleotides at the cleaved end that are not hydrogen bonded to another nucleotide on the opposite strand. In some embodiments, a double-strand break results in a "sticky" end of a double-stranded polynucleotide, wherein the cleavage is performed between nucleotides at different relative positions on each strand of the two strands, resulting in a polynucleotide overhang on one strand compared to the other. When a first cleavage of a first polynucleotide backbone occurs between a first set of two nucleotides on one strand, and a second cleavage of a second polynucleotide backbone occurs between a second set of two nucleotides on the opposite strand, a "sticky" end shearing of a double-stranded polynucleotide is produced, wherein no more than one nucleotide of the first set hydrogen bonds with one of the nucleotides of the second set on the opposite strand, resulting in an "overhang" of at least one polynucleotide on one of the two strands, wherein the lengths of the two resulting sheared strands are not the same. In some embodiments, the DSB agent comprises more than one type of molecule. In a non-limiting example, the DSB agent comprises an endonuclease protein and a polynucleotide, such as a Cas endonuclease and a guide RNA. In some aspects, the DSB agent is a fusion protein comprising a plurality of polypeptides. In a non-limiting example, the DSB agent is a Cas endonuclease with an inactivated nuclease domain, and another polypeptide with nuclease activity.

[0162] As used herein, the term "recognition site" refers to a polynucleotide sequence to which a double-strand break inducing agent can be aligned and can optionally contact, bind and / or achieve double-strand breaks. The terms "recognition site" and "recognition sequence" are used interchangeably herein. The recognition site can be an endogenous site in the genome of a host (e.g., yeast, animal or plant), or alternatively, the recognition site can be heterologous to the host (yeast, animal or plant), and therefore is not naturally present in the genome, or can be found in a heterologous genomic position compared to the position present in nature. The length and composition of the recognition site can be unique or specific to the double-strand break inducing agent. The cleavage site of the DSB agent can be the same or different from the recognition site, and can be the same or different from the binding site.

[0163] As used herein, the term "endogenous recognition (or binding or cleavage) site" refers to a double-strand break-inducing agent recognition (or binding or cleavage) site that is endogenous or native to the host (e.g., plant, animal or yeast) genome and is located at the endogenous or native location of the recognition (or binding or cleavage) site in the genome of the host (e.g., plant, animal or yeast). 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, The composition of the recognition (or binding or cutting) site can vary, and includes, for example, a plurality of specific nucleotides whose composition is recognized by the DSB agent. In some aspects, these multiple specific nucleotides are continuous in the primary sequence. In some aspects, these multiple specific nucleotides are discontinuous in the primary sequence. It is also possible that the recognition site can be palindromic, that is, the sequence on one chain is identical to the reading in the opposite direction on the complementary chain. The binding and / or nick / cut site can be within the recognition sequence, or the binding and / or nick / cut site can be outside the recognition sequence. In another variation, DSB cutting can occur at nucleotide positions that are just opposite to each other to produce flat end cutting, or in other cases, the nick can be staggered to produce single-stranded overhangs, also referred to as "sticky ends", which can be 5' overhangs or 3' overhangs.

[0164] As used herein, the term "target recognition site" refers to a polynucleotide sequence to which a double-strand break inducing agent can perfectly align (ie, zero nucleotide mismatches, gaps, or insertions) and induce a double-strand break in some aspect.

[0165] As used herein, the term "target binding site" refers to a polynucleotide sequence at which a double-strand break-inducing agent is able to form a functional association at the polynucleotide sequence and form a bond with the complementary nucleotide of the target polynucleotide chain under perfect alignment (i.e., zero nucleotide mismatches, gaps, or insertions).

[0166] As used herein, the term "target cleavage site" refers to a polynucleotide sequence at which a double-strand break-inducing agent is capable of generating a double-strand break with perfect alignment (ie, zero nucleotide mismatches, gaps, or insertions) at the polynucleotide sequence.

[0167] "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats) loci refer to certain genetic loci encoding components of a DNA cleavage system, such as those used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327: 167-170; WO 2007025097 published March 1, 2007). A CRISPR locus may consist of a CRISPR array that may be flanked by different Cas (CRISPR associated) genes, comprising short direct repeat sequences (CRISPR repeats) separated by short variable DNA sequences (called 'spacers').

[0168] As used herein, "effector" or "effector protein" is a protein with an activity that includes recognition, binding, and / or cleavage or nicking of a polynucleotide target. The "effector complex" of the CRISPR system includes Cas proteins that participate in crRNA and target recognition and binding. Some component Cas proteins may additionally include domains that participate in target polynucleotide cleavage.

[0169] The term "Cas protein" refers to a polypeptide encoded by a Cas (CRISPR-related) gene. Cas proteins include, but are not limited to, novel Cas-δ proteins disclosed herein, Cas9 proteins, Cpf1 (Cas12) proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or a combination or complex of these. When complexed with a suitable polynucleotide component, the Cas protein can be a "Cas endonuclease" or "Cas effector protein" that can recognize, bind, and optionally nick or cut all or part of a specific polynucleotide target sequence. The Cas endonucleases described herein comprise one or more nuclease domains. The Cas-δ endonucleases disclosed herein may include those having a RuvC or RuvC-like nuclease domain. The Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a functional fragment or functional variant of a native Cas protein, or a functional fragment or functional variant of a native Cas protein having at least 50%, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 450, at least 500, or more than 500 consecutive amino acids having at least 50%, between 50% and 55%, at least 55%, 55% and 60% of the amino acids in the native Cas protein. %, at least 97%, 97% and 98%, at least 98%, 98% and 99%, at least 99%, 99% and 100%, or 100% sequence identity to the polypeptide or protein and retains at least partial activity.

[0170] The terms "cascade" and "cascade complex" are used interchangeably herein and include reference to a multi-subunit protein complex that can be assembled with a polynucleotide to form a polynucleotide-protein complex (PNP). A cascade is a polynucleotide-dependent PNP to achieve complex assembly and stability and to identify target nucleic acid sequences. The cascade is used as a monitoring complex that finds and optionally binds to a target nucleic acid that is complementary to the variable targeting domain of a guide polynucleotide.

[0171] The terms "cleavage-ready Cascade", "crCascade", "cleavage-ready Cascade complex", "crCascade complex", "cleavage-ready Cascade system", "CRC" and "crCascade system" are used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP), wherein one of the cascade proteins is a Cas endonuclease that is capable of recognizing, binding, and optionally unwinding, nicking or cleaving all or part of a target sequence.

[0172] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease (including a Cas endonuclease described herein) and enable the Cas endonuclease to recognize, optionally bind, and optionally cleave a DNA target site. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence).

[0173] The terms "single guide RNA" and "sgRNA" are used interchangeably herein and refer to the synthetic fusion of two RNA molecules, wherein a crRNA (CRISPR RNA) containing a variable targeting domain (connected to a tracr pairing sequence hybridized to tracrRNA) is fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA may comprise a crRNA or crRNA fragment of a type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease and a tracrRNA or tracrRNA fragment, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, such that the Cas endonuclease can recognize, optionally bind, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0174] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and include a nucleotide sequence that can hybridize (be complementary) to one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can 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%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a continuous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0175] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and include nucleotide sequences that interact with a Cas endonuclease polypeptide. A CER domain comprises a (trans-acting) tracr nucleotide pairing sequence followed by a tracr nucleotide sequence. A CER domain may be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., US20150059010 A1, published on February 26, 2015), or any combination thereof.

[0176] As used herein, the terms "guide polynucleotide / Cas endonuclease complex", "guide polynucleotide / Cas endonuclease system", "guide polynucleotide / Cas complex", "guide polynucleotide / Cas system" and "guided Cas system", "polynucleotide-guided endonuclease", "PGEN" are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease capable of forming a complex, wherein the guide polynucleotide / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site. The guide polynucleotide / Cas endonuclease complexes herein may comprise one or more Cas proteins and one or more suitable polynucleotide components of any known CRISPR system (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova et al., 2015, Nature Reviews Microbiology 13: 1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13).

[0177] 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 capable of forming a complex, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0178] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to the (targeted) target sequence (pre-spacer) recognized by the guide polynucleotide / Cas endonuclease system described herein. If the target DNA sequence is not followed by a PAM sequence, the Cas endonuclease may not successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0179] As used herein, "donor DNA" is a DNA construct comprising a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.

[0180] The term "polynucleotide modification template" includes a polynucleotide comprising at least one nucleotide modification when compared to a nucleotide sequence to be edited. The nucleotide modification may be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template may further comprise a homologous nucleotide sequence flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to the desired nucleotide sequence to be edited.

[0181] As used herein, "homologous recombination (HR)" includes the exchange of DNA fragments at homologous sites between two DNA molecules. The frequency of homologous recombination is affected by multiple factors. Different organisms vary in the amount of homologous recombination and the relative proportion of homology and non-homologous recombination. Usually, the length of the homologous region can affect the frequency of homologous recombination events: the longer the homologous region, the higher the frequency. The length of the homologous region required for observing homologous recombination also varies with species. In many cases, at least 5kb of homology has been utilized, but homologous recombination with a homology as low as 25bp-50bp has been observed. See, e.g., Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992) Mol Cell Biol 12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

[0182] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, progeny of seeds and plants. Plant cells include but are not limited to cells derived from: seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen and microspores." Plant elements" are intended to refer to whole plants or plant components, which may include differentiation and / or undifferentiated tissues, such as but not limited to plant tissues, parts and cell types. In one embodiment, plant elements are one of: whole plants, seedlings, meristematic tissues, basic tissues, vascular tissues, epithelial tissues, seeds, leaves, roots, buds, stems, flowers, fruits, runners, bulbs, tubers, corms, asexual terminal branches, buds, young shoots, tumor tissues, and various forms of cells and cultures (e.g., unicellular, protoplasts, embryos and callus). The term "plant organ" refers to plant tissues or a group of tissues that constitute different parts of the morphology and function of a plant. As used herein, "plant element" is a synonym for "part" of a plant, refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with the term "tissue" throughout the text. Similarly, "plant propagation element" is intended to generally refer to any plant part capable of creating other plants via sexual or asexual propagation of the plant, such as, but not limited to, seeds, seedlings, roots, buds, cuttings, scions, grafts, runners, bulbs, tubers, corms, asexual terminal branches or young shoots. Plant elements may be present in plants or in plant organs, tissue cultures or cell cultures.

[0183] "Progeny" includes any subsequent generation of an organism produced by sexual or asexual reproduction.

[0184] As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant calli, plant pieces, and intact plant cells in plants or plant parts (such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, spikes, cobs, shells, stems, roots, root tips, anthers, etc.), together with these parts themselves. Grains mean mature seeds produced by commercial growers for purposes other than cultivation or propagation of species. Progeny, variants, and mutants of these regenerated plants are also included within the scope of the present invention, provided that these parts contain introduced polynucleotides.

[0185] The term "monocotyledonous" or "monocotyledonous plant" refers to a subclass of angiosperms, also known as the "monocots," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0186] The term "dicotyledonous" or "dicots" refers to a subclass of angiosperms, also known as the "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0187] In the context of the present disclosure, the term "hybridized" or "cross" means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses sexual crosses (one plant being pollinated by another plant) and selfing (self-pollination, i.e., when the pollen and ovules (or microspores and megaspores) are from the same plant or genetically identical plants).

[0188] The term "infiltration" refers to the phenomenon that the allele of the expectation of the genetic locus is transferred to another genetic background from a genetic background. For example, the allele infiltration of the expectation at the specified locus can be passed to at least one progeny plant via the sexual hybridization between two parental plants, wherein at least one of the parental plants has the allele of expectation in its genome. Alternatively, for example, the transmission of alleles can occur by the recombination between two donor genomes, such as in fusion protoplasts, wherein at least one of the donor protoplasts has the allele of expectation in its genome. The allele of expectation can be, for example, the allele of the selection of transgenic, modified (mutated or edited) natural allele or marker or QTL.

[0189] The term "isoline" is a comparative term referring to organisms that are genetically identical but different in treatment. In one example, two genetically identical maize plant embryos can be divided into two different groups, one group receiving treatment (such as introduction of CRISPR-Cas effector endonucleases), and one group not receiving such treatment as a control. Therefore, any phenotypic differences between the two groups may be attributed only to the treatment, rather than to any inherent nature of the endogenous gene composition of the plant.

[0190] The compositions and methods herein can provide plants with improved "agronomic traits" or "traits of agronomic importance" or "traits of agronomic significance", which traits can include, but are not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, improved yield, enhanced health, improved vigor, improved growth, improved photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root structure, modulation of metabolites, modulation of the proteome, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition compared to a homologous plant not comprising the modifications derived from the methods and compositions herein.

[0191] "Agronomic potential" is intended to mean the ability of a plant element to express a phenotype, preferably an improved agronomic trait, at some point in its life cycle, or to transmit said phenotype to another plant element with which it is associated in the same plant.

[0192] As used herein, the terms "reduced," "less," "slower," and "increased," "faster," "enhanced," "greater" refer to a decrease or increase in a characteristic of a modified plant element or resulting plant as compared to an unmodified plant element or resulting plant. For example, the reduction in a characteristic can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400%, %, or more, and the increase can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, 100% and 200%, at least 200%, at least about 300%, at least about 400%, or more above a control.

[0193] The meanings of the abbreviations are as follows: “sec” means second, “min” means minute, “h” means hour, “d” means day, “μL” means microliter, “mL” means milliliter, “L” means liter, “μM” means micromole, “mM” means millimolar, “M” means molar, “mmol” means millimole, “μmole” or “umole” means micromole, “g” means gram, “μg” or “ug” means microgram, “ng” means nanogram, “U” means unit, “bp” means base pair, and “kb” means kilobase.

[0194] Compositions and methods for modifying the genome of a target cell

[0195] In some aspects, methods and compositions for engineering site-specific promoter activation complexes near non-functional promoters are provided. In certain embodiments of this aspect, genomes are edited using the application of methods and compositions for engineering site-specific promoter activation complexes near non-functional promoters. For example, donor polynucleotides can be inserted into the genome and selected using the methods and compositions provided herein.

[0196] In other aspects, the site-specific promoter activation complex engineered near the non-functional promoter results in a higher level of expression of the non-functional promoter. In certain embodiments, the non-functional promoter comprises any polynucleotide sequence upstream of the coding sequence. The length and composition of the polynucleotide sequence include a non-functional promoter comprising a polynucleotide sequence of at least 10,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 9,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 8,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 7,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 6,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 5,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 4,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 3,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 2,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 1,000bp, a non-functional promoter comprising a polynucleotide sequence of at least 900bp, a non-functional promoter comprising at least 8 The non-functional promoter of the polynucleotide sequence of at least 100bp, the non-functional promoter comprising the polynucleotide sequence of at least 700bp, the non-functional promoter comprising the polynucleotide sequence of at least 600bp, the non-functional promoter comprising the polynucleotide sequence of at least 500bp, the non-functional promoter comprising the polynucleotide sequence of at least 400bp, the non-functional promoter comprising the polynucleotide sequence of at least 300bp, the non-functional promoter comprising the polynucleotide sequence of at least 200bp, the non-functional promoter comprising the polynucleotide sequence of at least 100bp, the non-functional promoter comprising the polynucleotide sequence of at least 50bp, the non-functional promoter comprising the polynucleotide sequence of at least 40bp, the non-functional promoter comprising the polynucleotide sequence of at least 30bp, the non-functional promoter comprising the polynucleotide sequence of at least 20bp, or the non-functional promoter comprising the polynucleotide sequence of at least 10bp. It will be understood by those skilled in the art that it is expected that such non-functional promoter will not drive the expression of the coding sequence operably connected to the downstream of the minimal promoter. Furthermore, if such a polynucleotide sequence does drive expression of a coding sequence operably linked downstream of the minimal promoter, such a minimal promoter will function to drive expression of the coding sequence at a low level.

[0197] In other embodiments, the non-functional promoter comprises a minimal promoter. The example of a minimal promoter includes a polynucleotide sequence of the truncation of a full-length promoter. Compared with a full-length promoter, such a minimal promoter drives a lower level of expression. In some aspects, a minimal promoter will include at least one promoter element. Non-limiting examples of such promoter elements include a TATA box, a CAAT box (also described as a CCAAT box), a transcription initiation site, an RNA polymerase binding site, or any combination thereof. A minimal promoter includes promoter elements necessary for RNA polymerase binding and transcription initiation. For RNA polymerase II promoters, promoters are identified by a TATA homology sequence motif of about 20 to 50 base pairs upstream of the transcription initiation site and a CAAT homology sequence motif of about 50 to 120 base pairs upstream of the transcription initiation site. The TATA motif is a site where the TATA binding protein (TBP) as a part of a complex (TFIID complex) of several polypeptides binds to factors that bind to other sequence elements of the promoter and effectively interacts (directly or indirectly). The TFIID complex then recruits the RNA polymerase II complex for transcription initiation, which is usually to be positioned at 25 to 30 base pairs downstream of the TATA element, and promotes extension, thereby producing RNA molecules. The sequences around the transcription initiation (designated as INR) of some polII genes seem to provide alternative binding sites for the members of the TFIID complex and thus the factors that "activate" transcription. These INR sequences are particularly relevant to promoters lacking functional TATA elements, which provide core promoter binding sites for final transcription. It has been proposed that promoters containing both functional TATA and INR motifs are the most effective in transcriptional activity. (Zenzie-Gregory et al., 1992. J. Biol. Chem. [Journal of Biological Chemistry] 267: 2823-2830). It is understood by those skilled in the art that elements other than the TATA motif are necessary for accurate transcription. Such elements are usually located upstream of the TATA motif, and a subset can have homology with the consensus sequence CCAAT. An exemplary minimal promoter suitable for use in plants is the minimal promoter of the ubiquitin-1 gene as disclosed in U.S. Patent Application No. 20130254943A1. An exemplary minimal promoter suitable for use in plants is the minimal promoter of the Arabidopsis thaliana ubiquitin-10 gene or the minimal promoter cassava vein mosaic virus promoter as disclosed in U.S. Patent Application No. 20160130595A1. An exemplary minimal promoter suitable for use in plants is the truncated CaMV 35S promoter.

[0198] In other embodiments, a known full-length plant promoter can be modified to be a non-functional promoter. It will be appreciated by those skilled in the art that molecular engineering of a polynucleotide sequence can result in the inactivation of a functional promoter, thereby rendering the promoter a non-functional promoter. Any of a variety of manipulations can be utilized, non-limiting examples of which include truncating a known plant promoter to produce a non-functional promoter, inverting the sequence of a known plant promoter to produce a non-functional promoter, or performing any other type of rearrangement of a known plant promoter's polynucleotide sequence to produce a non-functional promoter.

[0199] The known full-length plant promoters that can be modified as non-functional promoters include tissue-specific promoters. Tissue-specific promoters preferentially start transcription in certain tissues (e.g., stamens, anthers, filaments, and pollen) or developmental growth stages (e.g., spore-producing tissues, microspores, and microgametophytes). Such plant promoters are referred to as "tissue-preferred," "cell-type-preferred," or "growth-stage-preferred." Promoters that only start transcription in certain tissues are referred to as "tissue-specific." Similarly, promoters that only start transcription in certain growth stages are referred to as "growth-stage-specific." "Cell-type-specific" promoters drive expression only in certain cell types (e.g., stamen cells, or single cell types in stamens, such as anthers, filaments, or pollen cells) in one or more organs.

[0200] Known full-length plant promoters that can be modified to be non-functional promoters include male reproductive tissue-preferred promoters or tissue-specific promoters. One such promoter is the 5126 promoter, which preferentially directs expression of polynucleotides linked to male tissues of plants, as described in U.S. Pat. Nos. 5,837,851 and 5,689,051.Other examples include the maize Ms45 promoter described in U.S. Pat. No. 6,037,523; the SF3 promoter described in U.S. Pat. No. 6,452,069; the BS92-7 promoter described in WO 02 / 063021; ​​the SGB6 regulatory element described in U.S. Pat. No. 5,470,359; the TA29 promoter (Koltunow et al., (1990) Plant Cell 2:1201-1224; Nature 347:737 (1990); Goldberg, et al., (1993) Plant Cell 5:1217-1229 and U.S. Pat. No. 6,399,856); the SB200 gene promoter (WO 2002 / 26789), the PG47 gene promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No. 5,545,546; Plant Cell 5:1217-1229 and U.S. Pat. No. 6,399,856). J. Plant. 3(2):261-271 (1993)), G9 gene promoter (U.S. Pat. Nos. 5,837,850; 5,589,610); type 2 metallothionein-like gene promoter (Charbonnel-Campaa et al., Gene (2000) 254:199-208); Brassica Bca9 promoter (Lee et al., (2003) Plant Cell Rep. [Plant Cell Reports] 22:268-273); ZM13 promoter (Hamilton, et al., (1998) Plant Mol. Biol. 38:663-669); promoters of actin depolymerizing factors (e.g., Zmabp1, Zmabp2; see, e.g., Lopez et al., (1996) Proc. Natl. Acad. Sci. USA 93:7415-7420); promoter of maize pectin methylesterase-like gene ZmC5 (Wakeley et al., (1998) Plant Mol. Biol. 37:187-192); promoter of prefibrillarin gene Zmpro1 (Kovar et al., (2000) The Plant Cell 12:583-598); sulfated pentapeptide phytosulphokine gene ZmPSK1 (Lorbiecke et al., (2005) Journal of Experimental Botany [Journal of Experimental Botany] 56(417):1805-1819); the promoter of the calmodulin binding protein Mpcbp (Reddy, et al., (2000) J. Biol. Chem. [Journal of Biological Chemistry] 275(45):35457-70).

[0201] Known full-length plant promoters that can be modified to be non-functional promoters include constitutive promoters. Constitutive promoters include, for example, promoters of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Pat. No. 6,072,050; CaMV 35S promoter (Odell et al., (1985) Nature 313:810-812); rice actin (McElroy et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen et al., (1992) Plant Mol. Biol. 13:614-633; Mol. Biol. [Plant Molecular Biology] 18: 675-689); pEMU (Last et al., (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81: 581-588); MAS (Velten et al., (1984) EMBO J. [Journal of the European Molecular Biology Association] 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), etc. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0202] Known full-length plant promoters that can be modified to non-functional promoters include seed-preferred promoters. "Seed-preferred" promoters include those that are active during seed development (e.g., promoters of seed storage proteins) and those that are active during seed germination. See, Thompson et al. (1989) BioEssays [Biological Assays] 10: 108, which is incorporated herein by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19kDa zein); milps (inositol-1-phosphate synthase) (see WO00 / 11177 and U.S. Patent No. 6,225,529; incorporated herein by reference). γ-zein is an endosperm-specific promoter. Globulin-1 (Glob-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, etc. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, γ-zein, wax protein, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, which discloses seed-preferred promoters from end1 and end2 genes. Additional embryo-specific promoters are disclosed in Sato et al. (1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase et al. (1997) Plant J 12:235-46; and Postma-Haarsma et al. (1999) Plant Mol. Biol. 39:257-71. Additional endosperm-specific promoters are disclosed in Albani et al. (1984) EMBO [Journal of the European Molecular Biology Association] 3:1405-15; Albani et al. (1999) Theor. Appl. Gen. [Theoretical and Applied Genetics] 98:1253-62; Albani et al. (1993) Plant J. [Plant Journal] 4:343-55; Mena et al. (1998) The Plant Journal [Plant Journal] 116:53-62, and Wu et al. (1998) Plant Cell Physiology [Plant and Cell Physiology] 39:885-889.

[0203] Known full-length plant promoters that can be modified to be non-functional promoters include mitotic cell or meristem-preferred promoters. MITOTOX or meristem-preferred promoters are disclosed in Ito et al. (1994) Plant Mol. Biol. [Plant Molecular Biology] 24:863-878; Reyad et al. (1995) Mo. Gen. Genet. [Molecular Genetics and General Genetics] 248:703-711; Shaul et al. (1996) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 93:4868-4872; Ito et al. (1997) Plant J. [Plant Journal] 11:983-992; and Trehin et al. (1997) Plant Mol. Biol. [Plant Molecular Biology] 35:667-672.

[0204] Known full-length plant promoters that can be modified to be non-functional promoters include stress-inducible promoters. Stress-inducible promoters include salt / water stress-inducible promoters, such as P5CS (Zang et al. (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as cor15a (Hajela et al. (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm et al. (1993) Plant Mol Biol. 23:1073-1077), wsc120 (Ouellet et al. (1998) FEBS Lett. 423-324-328), ci7 (Kirch et al. (1997) Plant Mol Biol. 33:897-909), ci21A (Schneider et al. (1997) Plant Mol Biol. 33:897-909), Physiol. 113:335-45); drought-inducible promoters, for example, Trg-31 (Chaudhary et al. (1996) Plant Mol. Biol. 30:1247-57), rd29 (Kasuga et al. (1999) Nature Biotechnology 18:287-291); osmotic-inducible promoters, for example, Rab17 (Vilardell et al. (1991) Plant Mol. Biol. 17:985-93) and osmotic regulator (Raghothama et al. (1993) Plant Mol. Biol. 23:1117-28); and heat-inducible promoters, for example, heat shock proteins (Barros et al. (1992) Plant Mol. [Plant Molecules] 19:665-75; Marrs et al. (1993) Dev. Genet. [Genes and Development] 14:27-41) and smHSP (Waters et al. (1996) J. Experimental Botany [Experimental Botany] 47:325-338). Other stress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808 and U.S. Publication No. 2003 / 0217393) and rd29A (Yamaguchi-Shinozaki et al. (1993) Mol. Gen. Genetics [Molecular Genetics and General Genetics] 236:331-340).

[0205] In other aspects, the engineering of site-specific promoter activation complex near the non-functional promoter leads to the higher level expression of the non-functional promoter. In certain embodiments, the non-functional promoter drives the low-level expression of the operably connected coding sequence. In its aspect, the low-level expression leads to the generation of functional end products (for example, mRNA, guide RNA or protein), which can not be detected by the established molecular detection method, or such molecular detection method can be used to detect under low basic level. In other aspects, the low-level expression leads to the generation of functional end products (for example, mRNA, guide RNA or protein) that do not produce any biological effect to plant cells. As a non-limiting example, when treating cells with antibiotics or herbicides, the low-level expression of the coding sequence encoding antibiotic or herbicide selectable markers will not protect cells from damage or death. In other aspects, the low-level expression leads to not producing any functional end products (for example, mRNA, guide RNA or protein).

[0206] In another aspect, the proximity of the site-specific promoter activation complex to the non-functional promoter results in a higher level of expression of the non-functional promoter. In an embodiment, the site-specific promoter activation complex is designed to bind to the upstream polynucleotide sequence of the non-functional promoter. The site-specific promoter activation complex can be located at least 1 bp, at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 125 bp, at least 150 bp, at least 175 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 900 bp, at least 100 bp, at least 125 bp, at least 150 bp, at least 175 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least bp, at least 850bp, at least 900bp, at least 950bp, at least 1000bp, at least 1100bp, at least 1200bp, at least 1300bp, at least 1400bp, at least 1500bp, at least 2000bp, at least 2500bp, at least 3000bp, at least 3500bp, at least 4000bp, at least 4500bp, at least 5000bp, at least 5500bp, at least 6000bp, at least 6500bp, at least 7500bp, at least 8000bp, at least 8500bp, at least 9000bp, at least 9500bp or at least 10,000bp. Ideally, the spacing proximity is determined based on the highest expression level of the coding sequence.

[0207] In other aspects, the engineering of site-specific promoter activation complex near the non-functional promoter leads to the higher level expression of the non-functional promoter. In turn, the expression of the non-functional promoter increases and drives the expression of the coding sequence. In some aspects, high-level expression leads to the generation of functional end products (for example, mRNA, guide RNA or protein) that can be detected by established molecular detection methods. In other aspects, high-level expression leads to the generation of functional end products (for example, mRNA, guide RNA or protein) that produce measurable or observable biological effects in plant cells. As a non-limiting example, when cells are treated with antibiotics or herbicides, the high-level expression of the coding sequence encoding antibiotic or herbicide selectable markers will protect cells from damage or death. In other aspects, the site-specific promoter activation complex drives the expression of a non-functional promoter to be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% or 100%. In other aspects, the site-specific promoter activation complex drives the expression of a non-functional promoter to be at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times or at least 50 times. In some aspects, the site-specific promoter activation complex drives the expression of the coding sequence to be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% or 100%. In other aspects, the site-specific promoter activation complex drives the expression of the coding sequence to be at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times or at least 50 times.

[0208] In other aspects, the engineering of site-specific promoter activation complex near the non-functional promoter leads to the higher level expression of the non-functional promoter. In turn, the expression of the non-functional promoter increases and drives the expression of the coding sequence. In some aspects, the site-specific promoter activation complex is combined with the genomic DNA upstream of the non-functional promoter. On the other hand, the site-specific promoter activation complex is combined with the genomic DNA downstream of the non-functional promoter. In some aspects, multiple copies of the site-specific promoter activation complex are combined with the genomic DNA upstream of the non-functional promoter. Therefore, there can be 1,2,3,4,5,6,7,8,9,10 or more site-specific promoter activation complexes, which are combined with the genomic DNA upstream of the non-functional promoter. On the other hand, the site-specific promoter activation complex uses a covalent bond, a hydrogen bond or a key utilizing van der Waals force to combine with the genomic DNA downstream of the non-functional promoter.

[0209] site-specific promoter activation complex

[0210] In other aspects, the site-specific promoter activation complex comprises at least one activation domain operably connected to a site-specific binding protein. The activation domain can be fused to the site-specific binding protein by various methods known to those skilled in the art. For example, the polynucleotide coding sequence encoding the site-specific binding protein can be engineered to include the coding sequence of the activation domain. The chimeric molecule includes the coding sequence of the site-specific binding protein in the same frame as the coding sequence. After the coding sequence is translated, a chimeric protein comprising the site-specific binding protein operably connected to the activation domain is generated, thereby generating the site-specific promoter activation complex.

[0211] For example, the VP16 activation domain can be fused to the C-terminus of the inactivated Cas9 molecule. Another example is to fuse four tandem repeats of the VP16 activation domain to the Cas9 molecule to produce an inactivated Cas9-VP64 molecule. Other site-specific promoter activation complexes are known to those skilled in the art. As a non-limiting example, the art describes the fusion of inactivated Cas9 site-specific binding proteins with P65 activation domains, EDLL activation domains, TAL activation domains, and CBF1 activation domains. Other site-specific binding proteins have been operably linked to the activation domain. On the other hand, zinc fingers, TALENs, and meganucleases can be operably linked to the activation domain. In another aspect, the activation domain is operably linked to a site-specific binding protein to produce a site-specific promoter activation complex, wherein the activation domain is selected from RTA, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, VP64, VP16, VP160, GAL4, EDLL, ERF2, CBF1, ORCA2, DREB1A, LEAFY or any combination thereof.

[0212] When generating a site-specific promoter activation complex, multiple activation domains can be engineered into a site-specific binding protein. In some embodiments, the site-specific promoter activation complex comprises at least one activation domain, at least two activation domains, at least three activation domains, at least four activation domains, at least five activation domains, at least six activation domains, at least seven activation domains, at least eight activation domains, at least nine activation domains, at least ten activation domains, or ten or more activation domains that are bound to a site-specific binding protein.

[0213] In some aspects, the activation domain is operably connected to the site-specific binding protein as a continuous open reading frame. In an embodiment, by directly fusing the activation domain to the site-specific binding protein, the activation domain is operably connected to the site-specific binding protein. In an embodiment, by directly engineering the activation domain in the coding sequence of the site-specific binding protein, the activation domain is operably connected to the site-specific binding protein. In an embodiment, the activation domain is operably connected to the site-specific binding protein by the polynucleotide linker of the activation domain and the site-specific binding protein. In other embodiments, the activation domain is operably connected to the site-specific binding protein by epitope / antibody interaction (e.g., using the Sun Tag system).

[0214] Activation domain

[0215] In other aspects, the engineering of site-specific promoter activation complexes includes at least one activation domain. The activation domain is a protein that recruits many different proteins (e.g., nucleosome remodeling complexes; mediator complexes; and universal transcription factors, such as TFIIB, TBP, and TFIIH) involved in DNA transcription through protein-protein interactions to initiate or enhance transcription rate by affecting nucleosome assembly / disassembly, pre-initiation complex formation, promoter removal, and / or extension rate. The protein-protein interactions of transactivators and their binding partners involve discrete internal structural elements called "transactivation domains (TADs)" in transactivators. It is believed that TADs have almost no primary sequence homology and only adopt a limited structure when combined with a target. Sigler (1988) Nature [Nature] 333: 210-2. Although the acidic and hydrophobic residues in TADs are considered to be important (see, e.g., Cress and Triezenberg (1991) Science [Science] 251 (4989): 87-90), the contribution of a single residue to activity is considered to be small. Hall and Struhl (2002) J Biol. Chem. 277:46043-50.

[0216] Various activation domains are known in the art. Typically, the activation domain recruits the preinitiation complex to the promoter, and for the purposes of the present disclosure, recruits to a non-functional promoter sequence. There are many types of activation domains known in the art. And, these activation domains can be obtained from viruses, plants, animals or fungi. In some aspects, the activation domain is a transcription factor. Regardless of the source, the activation domain works to drive robust expression. In other aspects, the activation domain is selected from RTA, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, VP64, VP16, VP160, GAL4, EDLL, ERF2, CBF1, ORCA2, DREB1A, LEAFY or any combination thereof. Other examples of activation domains for use in the present disclosure include those provided in WO 2021178162A1 (incorporated herein in its entirety by reference), WO 2018183878 A1 (incorporated herein in its entirety by reference), and WO 2013116731 ​​A1 (incorporated herein in its entirety by reference). In further aspects, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more activation domains engineered with site-specific binding proteins to comprise site-specific promoter activation complexes.

[0217] site-specific binding protein

[0218] In other aspects, the engineering of site-specific promoter activation complexes includes site-specific binding proteins. In some applications in this regard, site-specific proteins include CRISPR, zinc finger proteins, TALEN proteins, or meganuclease proteins. In other aspects, CRISPR, zinc finger proteins, TALEN promoters, or meganuclease promoters are modified to bind DNA and do not cut or break the phosphodiester bonds of DNA. In some aspects, CRISPR, zinc finger proteins, TALEN proteins, or meganuclease proteins are mutated to bind DNA and do not cut or break the phosphodiester bonds of DNA. Such site-specific binding proteins include catalytically inactivated CRISPR or catalytically inactivated TALEN.

[0219] Zinc finger

[0220] For example, a zinc finger nuclease-mediated genome editing process is used to generate a genetically modified cell or plant as described herein. The process for editing a chromosome sequence includes, for example: (a) introducing the following into a cell: at least one nucleic acid encoding a zinc finger nuclease that recognizes a target sequence in a chromosome sequence and is capable of cutting a site in the chromosome sequence, and optionally, (i) at least one donor polynucleotide comprising a sequence for integration flanked by an upstream sequence and a downstream sequence that exhibits basic sequence identity to either side of the cleavage site, or (ii) at least one exchange polynucleotide comprising a sequence substantially identical to a portion of the chromosome sequence at the cleavage site and further comprising at least one nucleotide change; and (b) culturing cells to allow expression of zinc finger nucleases so that zinc finger nucleases introduce double-strand breaks into the chromosome sequence, and wherein the double-strand breaks are repaired by (i) a non-homologous end joining repair process so that an inactivating mutation is introduced into the chromosome sequence, or (ii) a homology-directed repair process so that the sequence in the donor polynucleotide is integrated into the chromosome sequence, or the sequence in the exchange polynucleotide is exchanged with a portion of the chromosome sequence. Zinc finger nucleases include a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease). Nucleic acids encoding zinc finger nucleases may include DNA or RNA. Zinc finger binding domains may be engineered to recognize and bind to any selected nucleic acid sequence. See, e.g., Beerli et al., (2002) Nat. Biotechnol. 20:135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416; and Doyon et al., (2008) Nat. Biotechnol. 26:702-708; Santiago et al., (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814; Urnov et al., (2010) Nat. Rev Genet. [Nature Review of Genetics] 11(9):636-46; and Shukla et al., (2009) Nature [Nature] 459(7245):437-41. Engineered zinc finger binding domains may have new binding specificities compared to naturally occurring zinc finger proteins. For example, the algorithm described in U.S. Pat. No. 6,453,242 can be used to design zinc finger binding domains to target preselected sequences.Non-degenerate recognition code tables can also be used to design zinc finger binding domains to target specific sequences (Sera et al., (2002) Biochemistry 41:7074-7081). Tools for identifying potential target sites in DNA sequences and designing zinc finger binding domains can be used (Mandell et al., (2006) Nuc. Acid Res. 34:W516-W523; Sande et al., (2007) Nuc. Acid Res. 35:W599-W605).

[0221] Exemplary zinc finger DNA binding domains recognize and bind to a sequence having at least about 80% sequence identity to a desired target sequence. In other embodiments, the sequence identity may be about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0222] Zinc finger nucleases also include a cleavage domain. The cleavage domain portion of a zinc finger nuclease can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2010-2011 Catalog, New England Biolabs, Beverly, Massachusetts; and Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388. Other enzymes known to cleave DNA (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). One or more of these enzymes (or their functional fragments) can be used as a source of a cleavage domain.

[0223] Meganuclease

[0224] Another example of genetically modifying the cells or plants described herein is to modify the plant genome by using a "custom" meganuclease that is generated (see, e.g., WO 2009 / 114321; Gao et al. (2010) Plant Journal 1: 176-187). The term "meganuclease" generally refers to a naturally occurring homing endonuclease that binds to double-stranded DNA at a recognition sequence that is greater than 12 base pairs and encompasses the corresponding intron insertion site. A naturally occurring meganuclease can be a monomer (e.g., I-SceI) or a dimer (e.g., I-CreI). As used herein, the term meganuclease can be used to refer to a monomeric meganuclease, a dimeric meganuclease, or a monomer that binds to form a dimeric meganuclease.

[0225] Naturally occurring meganucleases, such as those from the LAGLIDADG family, have been used to effectively promote site-specific genome modification in plants, yeast, fruit flies, mammalian cells and mice. Engineered meganucleases are known, such as the LIG-34 meganuclease, which recognizes and cleaves a 22 base pair DNA sequence found in the Zea mays (maize) genome (see, e.g., US20110113509).

[0226] TALEN

[0227] TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcription activators in the plant cell nucleus, where they bind directly to DNA via the central domain of tandem repeats. Transcription activator-like (TAL) effector DNA modification enzymes (TALE or TALEN) are also used for engineering genetic changes. See, for example, US20110145940, Boch et al., (2009), Science 326 (5959): 1509-12. Fusion of TAL effectors with FokI nuclease provides TALENs that bind and cut DNA at specific locations. Target specificity is determined by developing customized amino acid repeat sequences in TAL effectors.

[0228] CRISPR system components

[0229] Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains and include restriction endonucleases that cleave DNA at specific sites without damaging bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas ( C RISPR- associetal) effector endonucleases.

[0230] Cas endonucleases (as single effector proteins or effector complexes with other components) unwind DNA duplexes at target sequences and optionally cut at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, crRNA or guide RNA) complexed with the Cas effector protein. If the correct protoplast adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence, such recognition and cutting of the target sequence by the Cas endonuclease typically occurs. Alternatively, the Cas endonucleases herein may lack DNA cutting or nicking activity, but when complexed with a suitable RNA component, can still specifically bind to the DNA target sequence. (See also U.S. Patent Application US20150082478, published on March 19, 2015, and US20150059010, published on February 26, 2015).

[0231] Cas endonucleases can occur as single effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0232] Cas endonucleases that have been described include, but are not limited to, for example: Cas3 (characteristic of 1 class, type I systems), Cas9 (characteristic of 2 class, type II systems), and Cas12 (Cpf1) (characteristic of 2 class, type V systems).

[0233] Cas endonucleases and effector proteins can be used for targeted genome editing (via single and multiple double-strand breaks and nicks) and targeted genome regulation (via tethering epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases and can serve as scaffolds for assembling polyprotein and nucleic acid complexes via RNA tethers (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963).

[0234] The Cas endonuclease, when complexed with a cognate guide RNA, recognizes, binds, and optionally nicks or cleaves the target polynucleotide.

[0235] The Cas endonucleases, effector proteins or functional fragments thereof used in the disclosed methods can be isolated from natural sources or recombinant sources, in which genetically modified host cells are modified to express nucleic acid sequences encoding proteins. Alternatively, cell-free protein expression systems can be used to produce Cas proteins or synthetically produce Cas proteins. Effector Cas nucleases can be isolated and introduced into heterologous cells, or can be modified from their native form to exhibit activity types or activity magnitudes different from their natural sources. Such modifications include, but are not limited to, fragments, variants, substitutions, deletions, and insertions.

[0236] Fragments and variants of Cas endonucleases and Cas effector proteins can be obtained via methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art, such as, but not limited to, WO 2013166113 published on November 7, 2013, WO 2016186953 published on November 24, 2016, and WO2016186946 published on November 24, 2016.

[0237] Cas endonucleases may include modified forms of Cas polypeptides. Modified forms of Cas polypeptides may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of Cas proteins. For example, in some cases, the modified form of the Cas protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (US20140068797 published on March 6, 2014). In some cases, the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically "inactivated Cas" or "inactivated Cas (dCas)". Inactivated Cas / inactivated Cas includes inactivated Cas endonucleases (dCas). Catalytically inactivated Cas effector proteins may be fused to heterologous sequences to induce or modify activity.

[0238] The Cas endonuclease can be part of a fusion protein that also comprises one or more heterologous protein domains (e.g., 1, 2, 3 or more domains other than the Cas protein). Such a fusion protein can comprise any additional protein sequence, and optionally a linker sequence between any two domains (e.g., between the Cas and the first heterologous domain). Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]); reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], β-galactosidase, β-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]); and domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity (e.g., VP16 or VP64), transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0239] The catalytically active and / or catalytically inactivated Cas endonucleases can be fused to heterologous sequences (US20140068797 disclosed on March 6, 2014). Suitable fusion partners include, but are not limited to providing active polypeptides (such as site-specific promoter activation complexes), which are indirectly increased by acting directly on the target DNA or on the polypeptides (such as, histones or other DNA-binding proteins) related to the target DNA to transcribe. Other suitable fusion partners include, but are not limited to providing methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, Su-su (SUMOylating) activity, de-Su-su (deSUMOylating) activity, ribosylation activity, deribosylation activity, myristoylation activity, or de-myristoylation activity polypeptide. Further suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcriptional regulators, etc.). Partially active or catalytically inactive Cas-α endonucleases can also be fused to another protein or domain, such as Clo51 or FokI nucleases, to produce double-strand breaks (Guilinger et al. Nature Biotechnology, Vol. 32, No. 6, June 2014).

[0240] A catalytically active or catalytically inactive Cas protein, such as the Cas-α protein described herein, can also be fused to a molecule that directs the editing of single or multiple bases in a polynucleotide sequence, such as a site-specific deaminase that can change nucleotide identity, such as from C·G to T·A or from A·T to G·C (Gaudelli et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305) (2016); Komor et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA." "Cleaving." [Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage.]" Nature 533(7603)(2016):420-4. The base editing fusion protein can include, for example, an active (double-strand break generation), partially active (nickase) or inactivated (catalytically inactive) Cas-α endonuclease and a deaminase (such as, but not limited to, cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, base editing repair inhibitors and glycosylase inhibitors (e.g., uracil glycosylase inhibitors (to prevent uracil removal)) are considered as other components of the base editing system.

[0241] Cas endonucleases can be expressed and purified by methods known in the art, for example as described in WO / 2016 / 186953 published on November 24, 2016.

[0242] Guide polynucleotide

[0243] Instruction polynucleotide enables Cas endonuclease to target recognition, binding and optionally cutting, and can be unimolecular or bimolecular.Instruction polynucleotide sequence can be RNA sequence, DNA sequence or its combination (RNA-DNA combination sequence). Optionally, instructing polynucleotide can include at least one nucleotide, phosphodiester bond or connection modification, such as but not limited to locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, thiophosphate bond, connection with cholesterol molecule, connection with polyethylene glycol molecule, connection with spacer 18 (hexaethylene glycol chain) molecule or cause cyclization 5' to 3' covalent connection. Instruction polynucleotides comprising only ribonucleic acid are also referred to as "guidance RNA" or "gRNA" (US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015). Instruction polynucleotide can be engineered or synthesized.

[0244] The guide polynucleotide includes a chimeric non-naturally occurring guide RNA that includes regions that are not found together in nature (i.e., they are heterologous to each other). For example, the chimeric non-naturally occurring guide RNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, and the first nucleotide sequence domain is connected to a second nucleotide sequence that can recognize a Cas endonuclease, such that the first and second nucleotide sequences are not found connected together in nature.

[0245] The guiding polynucleotide can be a double molecule (also referred to as a duplex guiding polynucleotide) comprising a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide (e.g., tracrRNA) sequence. In some cases, there is a linker polynucleotide connecting crRNA and tracrRNA to form a single guiding object (e.g., sgRNA).

[0246] The cr nucleotides include a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA and a C A second nucleotide sequence (also called a tracr mate sequence) that is part of the endonuclease recognition (CER) domain. The tracr mate sequence can hybridize to the tracr nucleotide along the complementary region and together form CAs endonuclease recognition domain or CER domain. The CER domain can interact with the Cas endonuclease polypeptide. The cr nucleotides and tracr nucleotides of the duplex guidance polynucleotide can be RNA, DNA and / or RNA-DNA combination sequences. In some embodiments, the cr nucleotide molecules of the duplex guidance polynucleotide are referred to as "crDNA" (when composed of a continuous extension of DNA nucleotides) or "crRNA" (when composed of a continuous extension of RNA nucleotides) or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides may be included in fragments of crRNA naturally occurring in bacteria and archaea. The size of the crRNA fragments naturally occurring in bacteria and archaea that may be present in the cr nucleotides disclosed herein may be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the crRNA molecule is selected from the group consisting of: SEQ ID NO: 57, 58 and 59.

[0247] In some embodiments, the tracr nucleotide is referred to as "tracrRNA" (when composed of a continuous extension of RNA nucleotides) or "tracrDNA" (when composed of a continuous extension of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. In the 5'- to -3' direction, tracrRNA (trans-activating CRISPR RNA) comprises (i) a sequence annealed to the repeat region of CRISPR II type crRNA and (ii) a portion containing a stem loop (Deltcheva et al., Nature [Nature] 471: 602-607). The duplex guide polynucleotide can form a complex with the Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to the target, and optionally nick or cut (introduce single-stranded or double-stranded breaks) the target site. (US 20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015).

[0248] The guide polynucleotide can also be a single molecule (also referred to as a single guide polynucleotide) comprising a cr nucleotide sequence linked to a tracr nucleotide sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA and a Cas endonuclease recognition domain (CER domain) that interacts with a Cas endonuclease polypeptide.

[0249] Protospacer adjacent motif (PAM)

[0250] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to a (targeted) target sequence (pre-spacer) that can be recognized by a guide polynucleotide / Cas endonuclease system. If the target DNA sequence is not followed by a PAM sequence, the Cas endonuclease may not be able to successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0251] "Randomized PAM" and "randomized protospacer adjacent motif" are used interchangeably herein and refer to a random DNA sequence adjacent to a target sequence (protospacer) recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The randomized 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 in length. The randomized nucleotides include any of the nucleotides A, C, G or T.

[0252] Guide polynucleotide / Cas endonuclease complex

[0253] The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding, and optionally nicking, unwinding, or cleaving all or part of a target sequence.

[0254] The guiding polynucleotide / Cas endonuclease complex that can cut both chains of the DNA target sequence typically comprises a Cas protein with all its endonuclease domains in a functional state (e.g., a wild-type endonuclease domain or a variant thereof that retains some or all activity in each endonuclease domain). Therefore, a wild-type Cas protein (e.g., a Cas protein disclosed herein) or a variant thereof that retains some or all activity in each endonuclease domain of the Cas protein is a suitable example of a Cas endonuclease that can cut both chains of the DNA target sequence.

[0255] The guiding polynucleotide / Cas endonuclease complex that can cut one strand of the DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleavage ability). The Cas nickase typically comprises a functional endonuclease domain that allows Cas to cut only one strand of the DNA target sequence (i.e., form a nick). For example, the Cas9 nickase can comprise (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, the Cas9 nickase can comprise (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in US20140189896, published on July 3, 2014. A pair of Cas nickases can be used to increase the specificity of DNA targeting. In general, this can be performed by providing two Cas nickases that target and nick near a DNA sequence in a desired targeted region on opposite strands by associating with RNA components having different guide sequences. Such near cleavage of each DNA strand generates a double-strand break (i.e., a DSB with a single-stranded overhang), which is then recognized as a substrate for non-homologous end joining (NHEJ) (which tends to produce incomplete repairs that lead to mutations) or homologous recombination (HR). Each nick in these embodiments can be separated from each other by, for example, at least about 5, 5 to 10, at least 10, 10 to 15, at least 15, 15 to 20, at least 20, 20 to 30, at least 30, 30 to 40, at least 40, 40 to 50, at least 50, 50 to 60, at least 60, 60 to 70, at least 70, 70 to 80, at least 80, 80 to 90, at least 90, 90 to 100 or 100 or more (or any integer between 5 and 100) bases. One or two Cas nickase proteins herein can be used for Cas nickase pairs. For example, a Cas9 nickase (i.e., Cas9HNH+ / RuvC-) having a mutated RuvC domain but having a functional HNH domain can be used (e.g., Streptococcus pyogenes Cas9HNH+ / RuvC-). By using the appropriate RNA components herein (which have guide RNA sequences that target each nickase to each specific DNA site), each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) is guided to specific DNA sites that are adjacent to each other (separated by up to 100 base pairs).

[0256] In certain embodiments, the polynucleotide / Cas endonuclease complex can bind to the DNA target site sequence, but does not cut any chain at the target site sequence. Such a complex can include a Cas protein in which all nuclease domains are mutated and dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence but does not cut any chain at the target site sequence can include a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. The Cas protein herein that binds but does not cut a target DNA sequence can be used to regulate gene expression, for example, in this case, the Cas protein can be fused to a transcription factor (or part thereof) (e.g., an inhibitor or activator, such as any of those disclosed herein).

[0257] Genome modification using novel CRISPR-Cas system components

[0258] As described herein, the guided Cas endonuclease can recognize, bind to the DNA target sequence, and introduce single-strand breaks (nicks) or double-strand breaks. Once single-strand breaks or double-strand breaks are induced in DNA, the DNA repair mechanism of the cell is activated to repair the break. Error-prone DNA repair mechanisms can produce mutations at double-strand break sites. The most common repair mechanism used to combine the ends of the break is the non-homologous end joining (NHEJ) pathway (Bleuyard et al., (2006) DNA Repair [DNA repair] 5: 1-12). The structural integrity of chromosomes is typically maintained by repair, but deletions, insertions or other rearrangements (such as chromosome translocations) are possible (Siebert and Puchta, 2002 Plant Cell [plant cells] 14: 1121-31; Pacher et al., 2007 Genetics [genetics] 175: 21-9).

[0259] DNA double-strand breaks appear to be potent factors in stimulating homologous recombination pathways (Puchta et al. (1995) Plant Mol Biol 28:281-92; Tzfira and White (2005) Trends Biotechnol 23:567-9; Puchta (2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- to nine-fold increase in homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al. (1995) Plant Mol Biol 28:281-92). Experiments with linear DNA molecules in maize protoplasts demonstrated enhanced homologous recombination between plasmids (Lyznik et al. (1991) Mol Gen Genet 230:209-18).

[0260] Homologous-directed repair (HDR) is a mechanism used to repair double-stranded DNA breaks and single-stranded DNA breaks in cells. Homologous directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber.2010Annu.Rev.Biochem.[Biological Chemistry Yearbook] 79:181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirement between donor and recipient DNA. Other forms of HDR include single-strand annealing (SSA) and break-induced replication, and these require shorter sequence homology relative to HR. Homologous directed repair at the nick (single-strand break) can occur via a mechanism different from HDR at the double-strand break (Davis and Maizels.PNAS[Proceedings of the National Academy of Sciences of the United States] (0027-8424), 111 (10), pages E924-E932).

[0261] The alteration of the genome of prokaryotic and eukaryotic cells or organism cells, for example by homologous recombination (HR), is a powerful tool for genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al., (1992) Mol Gen Genet [Molecular and General Genetics] 231: 186-93) and insects (Dray and Gloor, 1997, Genetics [Genetics] 147: 689-99). Homologous recombination can also be achieved in other organisms. For example, in the parasitic protozoan Leishmania, at least 150bp-200bp of homology is required for homologous recombination (Papadopoulou and Dumas, (1997) Nucleic Acids Res [Nucleic Acids Research] 25: 4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved with only 50 bp of flanking homology (Chaveroche et al., (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in the ciliate Tetrahymena thermophila (Gaertig et al., (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination has been most successful in mice using pluripotent embryonic stem (ES) stem cell lines that can be grown in culture, transformed, selected, and introduced into mouse embryos (Watson et al., (1992) Recombinant DNA, 2nd ed., Scientific American Books distributed by WH Freeman & Co.).

[0262] Gene targeting

[0263] The guidance polynucleotide / Cas system described herein can be used for gene targeting. Generally, DNA targeting can be carried out by cutting one or two chains at the specific polynucleotide sequence in the cell with the Cas protein associated with a suitable polynucleotide component. Once single-strand breaks or double-strand breaks are induced in DNA, the DNA repair mechanism of the cell is activated to repair the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes that can cause the modification at the target site.

[0264] The length of the DNA sequence at the target site can vary, and includes, for example, a target site of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 30 nucleotides in length. It is also possible that the target site can be palindromic, that is, the sequence on one strand is the same when read in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence, or the nick / cleavage site can be outside the target sequence. In another variation, the cleavage can occur at nucleotide positions that are exactly opposite to each other to produce a blunt-ended cleavage, or in other cases, the nicks can be staggered to produce single-stranded overhangs, also known as "sticky ends", which can be 5' overhangs or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants may comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a given target site, wherein these active variants retain biological activity and are therefore capable of being recognized and cleaved by a Cas endonuclease.

[0265] Assays that measure single-strand breaks or double-strand breaks at a target site caused by an endonuclease are known in the art, and generally measure the overall activity and specificity of the agent on a DNA substrate comprising the recognition site.

[0266] The targeting method herein can be carried out in such a way that two or more DNA target sites are targeted, for example, in the method. Such a method can be optionally characterized as a multiplexing method. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten or more target sites can be targeted simultaneously. The multiplexing method is typically carried out by the targeting method herein, wherein a plurality of different RNA components are provided, each of which is designed to guide the polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0267] Gene Editing

[0268] The process of editing a genomic sequence by combining DSB and modification templates generally includes: introducing a DSB inducer or a nucleic acid encoding a DSB inducer (which recognizes a target sequence in a chromosomal sequence and is capable of inducing a DSB in a genomic sequence) into a host cell, and at least one polynucleotide modification template comprising at least one nucleotide change when compared to the nucleotide sequence to be edited. The polynucleotide modification template may further comprise a nucleotide sequence flanking the at least one nucleotide change, wherein the flanking sequence is substantially homologous to the chromosomal region flanking the DSB. Genome editing using DSB inducers (such as Cas-gRNA complexes) has been described, for example, in US20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and WO / 2016 / 025131 published on February 18, 2016.

[0269] Some uses of the guide RNA / Cas endonuclease system have been described (see, for example: US20150082478 A1 published on March 19, 2015, WO 2015026886 published on February 26, 2015, and US20150059010 published on February 26, 2015) and include, but are not limited to, modification or replacement of a target nucleotide sequence (such as a regulatory element), insertion of a target polynucleotide, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a target protein, amino acid and / or protein fusion, and gene silencing caused by expression of an inverted repeat sequence in a target gene.

[0270] Proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of one or more proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance for amino acid substitutions that are unlikely to affect the biological activity of a protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC [National Biomedical Research Foundation, Washington, DC, USA]. Conservative substitutions, such as exchanging one amino acid for another with similar properties, would be preferred. Conservative deletions, insertions, and amino acid substitutions are expected to produce no radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by conventional screening assays. Assays for double-strand-break-inducing activity are known, and generally measure the overall activity and specificity of an agent for a DNA substrate comprising a target site.

[0271] Methods for genome editing with Cas endonucleases and complexes of Cas endonucleases and guide polynucleotides are described herein. After characterizing the guide RNA and PAM sequences, endonucleases and associated CRISPR RNA (crRNA) components can be used to modify chromosomal DNA in other organisms including plants. In order to promote optimal expression and nuclear localization (for eukaryotic cells), the complex containing the gene can be optimized as described in WO 2016186953 published on November 24, 2016, and then delivered to the cell as a DNA expression cassette by methods known in the art. The components that must contain the active complex can also be delivered as RNA (with or without modifications to protect RNA from degradation) or as capped or uncapped mRNA (Zhang, Y. et al., 2016, Nat. Commun. [Natural Communications] 7: 12617) or Cas protein guide polynucleotide complexes (published in WO 2017070032 on April 27, 2017), or any combination thereof. In addition, one or more parts of the complex and crRNA can be expressed from a DNA construct, while the other components are delivered as RNA (with or without modifications to protect the RNA from degradation) or as capped or uncapped mRNA (Zhang et al. 2016 Nat. Commun. [Natural Communications] 7: 12617) or Cas protein-guided polynucleotide complexes (published on April 27, 2017 WO 2017070032) or any combination thereof. For in vivo production of crRNA, tRNA-derived elements can also be used to recruit endogenous RNases to cut crRNA transcripts into mature forms that can guide the complex to its DNA target site, for example, as described in WO 2017105991 published on June 22, 2017. The nickase complex can be used alone or in combination to generate single or multiple DNA nicks on one or both DNA strands. In addition, the cleavage activity of Cas endonucleases can be inactivated by changing the key catalytic residues in the cleavage domain (Sinkunas, T. et al., 2013, EMBO J. [Journal of the European Molecular Biology Association] 32: 385-394), thereby generating an RNA-guided helicase that can be used to enhance homology-directed repair, induce transcriptional activation, or reshape local DNA structure. In addition, the activity of Cas cleavage and helicase domains can be knocked out and used in combination with other DNA shearing, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.

[0272] The transcription direction of tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domains, crRNA repeats, loops, anti-repeat sequences) can be derived as described in WO 2016186946 published on November 24, 2016 and WO 2016186953 published on November 24, 2016.

[0273] As described herein, once the appropriate guide RNA requirements are established, the PAM preference of each new system disclosed herein can be checked. If the cleavage complex results in degradation of the random PAM library, the complex can be converted into a nicking enzyme by mutagenesis of key residues or by assembling the reaction in the absence of ATP to disable the ATPase-dependent helicase activity, as previously described (Sinkunas, T. et al., 2013, EMBO J. [Journal of the European Molecular Biology Association] 32: 385-394). Double-stranded DNA breaks can be generated using two regions of PAM randomization separated by two pre-spacer targets, which can be captured and sequenced to check the PAM sequence that supports the cleavage of each complex.

[0274] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein into a cell, and identifying at least one cell having a modification at the target, wherein the modification at the target site is 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, chemical alteration of at least one nucleotide, and (v) any combination of (i)-(iv).

[0275] The nucleotide to be edited can be located inside or outside the target site recognized and cut by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification on the target site recognized and cut by the Cas endonuclease. In another embodiment, there are 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 at least one nucleotide to be edited and the genomic target site.

[0276] Knockouts can be produced by indels (insertion or deletion of nucleotide bases in the target DNA sequence by NHEJ), or by specific removal of sequences that reduce or completely abolish sequence function at or near the targeted site.

[0277] Guide polynucleotide / Cas endonuclease-induced targeted mutations can occur in nucleotide sequences located inside or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0278] The method for editing a nucleotide sequence in the genome of a cell may be a method by restoring the function of a non-functional gene product without using an exogenous selectable marker.

[0279] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein and at least one donor DNA into the cell, wherein the donor DNA comprises a polynucleotide of interest, and optionally, the method further comprises identifying at least one cell that has integrated the polynucleotide of interest into or near the target site.

[0280] In one aspect, the methods disclosed herein can employ homologous recombination (HR) to provide for integration of a polynucleotide of interest at a target site.

[0281] A variety of methods and compositions can be used to produce cells or organisms with a polynucleotide of interest inserted into a target site through the activity of a CRISPR-Cas system component as described herein. In one method described herein, a polynucleotide of interest is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" is a DNA construct comprising a polynucleotide of interest to be inserted into a target site of a Cas endonuclease. The donor DNA construct further comprises a first homology region and a second homology region flanking the polynucleotide of interest. The first homology region and the second homology region of the donor DNA have homology to the first and second genomic regions present in the target site of a cell or organism genome or flanking the target site, respectively.

[0282] Donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, can be used for genome editing, gene insertion and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963).

[0283] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or having a length between about 1 bp-20 bp, 20 bp-50 bp, 50 bp-100 bp, 75 bp-150 bp, 100 bp-250 bp, 150 bp-300 bp, 200 bp-400 bp, 250 bp-500 bp, 300 bp-600 bp, 350 bp-750 bp, 400 bp-800 bp, 450 bp- The target site can be a target site having a length of 1-20 bp, 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, and 20 bp. The amount of homology can also be described by the percentage sequence identity over the length of the complete alignment of two polynucleotides, which includes 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% percentage sequence identity. Sufficient homology includes any combination of polynucleotide length, overall percentage sequence identity, and optionally conserved regions of consecutive nucleotides or local percentage sequence identity, for example, sufficient homology can be described as a region of 75bp-150bp with at least 80% sequence identity to the region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, New York); 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).

[0284] Episomal DNA molecules can also be ligated into double-strand breaks, for example, T-DNA is integrated into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J. 17:6086-95). Once the sequence surrounding the double-strand break is altered, for example by mature exonuclease activity involved in double-strand breaks, the gene conversion pathway can restore the original structure if there are homologous sequences, such as homologous chromosomes in non-dividing somatic cells, or sister chromatids after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0285] In one embodiment, the present disclosure comprises a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one PGEN described herein and a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification of the nucleotide sequence, and the method optionally further comprises selecting at least one cell comprising the edited nucleotide sequence.

[0286] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of the target genomic nucleotide sequence. (See also US20150082478 published on March 19, 2015 and WO 2015026886 published on February 26, 2015).

[0287] The polynucleotides of interest and / or traits can be stacked together in a complex trait locus as described in WO 2012129373 published on September 27, 2012 and WO 2013112686 published on August 1, 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides an efficient system for generating double strand breaks and allows for the stacking of traits in a complex trait locus.

[0288] The guide polynucleotide / Cas system for mediating gene targeting as described herein can be used in the following methods for guiding heterologous gene insertion and / or generating complex trait loci comprising multiple heterologous genes in a manner similar to that disclosed in WO 2012129373 published on September 27, 2012, wherein the guide polynucleotide / Cas system as disclosed herein is used to replace the introduction of the target gene using a double-strand break inducing agent. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5 centimorgans (cM) of each other, these transgenes can be bred as a single genetic locus (e.g., see US20130263324 published on October 3, 2013 or WO2012129373 published on March 14, 2013). After selecting plants comprising transgenes, plants comprising (at least) one transgene can be crossed to form F1 comprising two transgenes. In the progeny from these F1 (F2 or BC1), 1 / 500 of the progeny will have two different transgenes recombined on the same chromosome. The composite locus can then be bred to a single genetic locus with two transgenic traits. This process can be repeated to stack as many traits as possible.

[0289] Further uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, US20150059010 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and PCT application WO2016025131 published on February 18, 2016) and include, but are not limited to, modification or replacement of a nucleotide sequence of interest (such as a regulatory element), insertion of a polynucleotide of interest, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing caused by expression of an inverted repeat sequence in a gene of interest.

[0290] The characteristics produced by the gene editing compositions and methods described herein can be evaluated. Chromosome intervals associated with the target phenotype or trait can be identified. Various methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are extended to cover markers that will be linked to genes controlling the target trait. In other words, the chromosomal interval is extended so that any marker located within the interval (including the end markers defining the boundaries of the interval) can be used as markers for specific traits. In one embodiment, the chromosomal interval contains at least one QTL, and in addition, more than one QTL can indeed be included. Multiple QTLs very close in the same interval can confuse the association of a specific marker with a specific QTL, because a marker can be shown to be linked to more than one QTL. On the contrary, for example, if two very close markers show co-segregation with the desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same QTL or identifies two different QTLs. The term "quantitative trait locus" or "QTL" refers to a DNA region associated with the differential expression of a quantitative phenotypic trait in at least one genetic background (e.g., in at least one breeding population). The region of the QTL encompasses or is closely linked to one or more genes that affect the trait under consideration. "Alleles of a QTL" can include multiple genes or other genetic factors, such as haplotypes, in a continuous genomic region or linkage group. The alleles of a QTL can represent haplotypes within a specified window, where the window is a continuous genomic region that can be defined and tracked with a set of one or more polymorphic markers. Haplotypes can be specified to be defined by the unique fingerprint of the alleles at each marker within the window.

[0291] Introducing CRISPR-Cas system components into cells

[0292] The methods described herein do not depend on a particular method for introducing a sequence into an organism or cell, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the organism. Introducing includes reference to incorporating a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated into the genome of the cell, and includes reference to nucleic acids, proteins, or polynucleotide-protein complexes (PGEN, RGEN) being provided transiently (directly) to the cell.

[0293] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated introduction, transfection, transduction, cell penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual hybridization, sexual breeding, and any combination thereof.

[0294] For example, guide polynucleotides (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) can be directly introduced into cells (transiently) as single-stranded or double-stranded polynucleotide molecules. Guide RNA (or crRNA + tracrRNA) can also be introduced into cells indirectly by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding guide RNA (or crRNA + tracrRNA), which is operably connected to a specific promoter capable of transcribing the guide RNA (crRNA + tracrRNA molecule) in the cell. Specific promoters can be, but are not limited to, RNA polymerase III promoters, which allow RNA transcription with precisely defined unmodified 5'- and 3'-ends (Ma et al., 2014, Mol. Ther. Nucleic Acids [Molecular Therapy - Nucleic Acids] 3: e161; DiCarlo et al., 2013, Nucleic Acids Res. [Nucleic Acids Research] 41: 4336-4343; WO 2015026887 published on February 26, 2015). Any promoter capable of transcribing the guide RNA in a cell can be used, and these promoters include heat shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the guide RNA.

[0295] Plant cells are distinct from animal cells (eg, human cells), fungal cells (eg, yeast cells), and protoplasts, including, for example, in that they contain a plant cell wall that can serve as a barrier to the delivery of components.

[0296] Delivery of Cas endonucleases, and / or guide RNAs, and / or ribonucleoprotein complexes, and / or polynucleotides encoding any one or more of the foregoing into plant cells can be achieved by methods known in the art, such as, but not limited to, rhizobium-mediated transformation (e.g., Agrobacterium, Ochrobactrum), particle-mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (e.g., protoplasts), electroporation, cell-penetrating peptides, or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery.

[0297] Cas endonucleases herein, such as Cas endonucleases described herein, can be introduced into cells by directly introducing the Cas polypeptide itself (referred to as direct delivery of Cas endonucleases), mRNA encoding Cas proteins, and / or guiding polynucleotide / Cas endonuclease complexes themselves, using any method known in the art. Cas endonucleases can also be introduced into cells indirectly by introducing recombinant DNA molecules encoding Cas endonucleases. Using any method known in the art, endonucleases can be introduced into cells instantaneously, or endonucleases can be incorporated into the genome of host cells. Cell penetrating peptides (CPPs) as described in WO 2016073433 disclosed on May 12, 2016 can be used to promote the uptake of endonucleases and / or guided polynucleotides into cells. Any promoter capable of expressing Cas endonucleases in cells can be used, and these promoters include heat shock / heat-inducible promoters operably linked to nucleotide sequences encoding Cas endonucleases.

[0298] Direct delivery of polynucleotide modified templates into plant cells can be achieved by particle-mediated delivery, and any other direct delivery method, such as but not limited to polyethylene glycol (PEG)-mediated protoplast transfection, whisker-mediated transformation, electroporation, particle bombardment, cell penetrating peptides or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery can be successfully used to deliver polynucleotide modified templates in eukaryotic cells (e.g., plant cells).

[0299] Donor DNA can be introduced by any means known in the art. Donor DNA can be provided by any transformation method known in the art (including, for example, Agrobacterium-mediated transformation or biolistic particle bombardment). Donor DNA can be transiently present in the cell, or can be introduced via a viral replicon. In the presence of Cas endonucleases and target sites, donor DNA is inserted into the genome of the transformed plant.

[0300] The direct delivery of any one of the guided Cas system components can be accompanied by the direct delivery (co-delivery) of other mRNAs that can promote the enrichment and / or visualization of cells receiving the guidance polynucleotide / Cas endonuclease complex components. For example, direct co-delivery of mRNAs encoding phenotypic markers (such as, but not limited to, transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell [Plant Cell] 12: 65-79)) can be achieved by restoring the function of non-functional gene products without using exogenous selectable markers to select and enrich cells, as described in WO2017070032 published on April 27, 2017.

[0301] Introducing the guide RNA / Cas endonuclease complex described herein (representing the cleavable complex described herein) into a cell includes introducing the components of the complex into the cell individually or in combination, the introduction being directly (direct delivery as RNA (for guides) and proteins (for Cas endonucleases and protein subunits or functional fragments thereof)) or via a recombinant construct expressing these components (guide RNA, Cas endonucleases, protein subunits or functional fragments thereof). Introducing the guide RNA / Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. The ribonucleotide-protein can be assembled prior to introduction into a cell as described herein. Components comprising guide RNA / Cas endonuclease ribonucleotide proteins (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled in vitro or by any method known in the art prior to introduction into a cell (the cell is targeted for genome modification as described herein).

[0302] The direct delivery of RGEN ribonucleoprotein allows genome editing at the target site in the genome of the cell, which can be followed by rapid degradation of the complex, and only allows the complex to exist briefly in the cell. This short-lived presence of the RGEN complex may cause the off-target effect to be reduced. In contrast, the delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can cause RGEN to be constantly expressed from these plasmids, and this constant expression can strengthen the off-target effect (Cradick, TJ et al. (2013) Nucleic Acids Res [Nucleic Acids Research] 41: 9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. [Natural Biotechnology] 31: 822-826).

[0303] Direct delivery can be achieved by combining any one of the components of a guide RNA / Cas endonuclease complex (RGEN) (standing for a cleavage-ready complex as described herein, such as at least one guide RNA, at least one Cas protein, and optionally at least one additional protein) with a delivery matrix comprising microparticles, such as but not limited to gold particles, tungsten particles, and silicon carbide whisker particles (see also WO 2017070032 published on April 27, 2017). The delivery matrix can comprise any of these components, such as a Cas endonuclease, attached to a solid matrix (e.g., a particle for bombardment).

[0304] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the site-specific promoter activation complex proteins that form the site-specific promoter activation complex are introduced into the cell as RNA and protein, respectively.

[0305] In one aspect, the site-specific promoter activation complex is a complex in which the activation domain and the site-specific binding protein that form the site-specific promoter activation complex and at least one protein subunit of the complex are introduced into the cell as RNA and protein, respectively.

[0306] In one aspect, the site-specific promoter activation complex is a complex in which an activation domain and a site-specific binding protein that form the site-specific promoter activation complex and at least one protein subunit of the complex are preassembled in vitro and introduced into a cell as a ribonucleotide-protein complex.

[0307] Protocols for introducing polynucleotides, polypeptides or polynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells such as plants or plant cells are known and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), whisker-mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide [Properties and Applications of Silicon Carbide] (2011), 345-358. Editor: Gerhardt, Rosario. Publisher: InTech, Rijeka, Croatia.CODEN: 69PQBP; ISBN: 978-953-307-201-2), direct gene transfer (Paszkowski et al., (1984) EMBO J [Journal of the European Molecular Biology Association] 3: 2717-22), and ballistic particle acceleration (U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods [Plant Cell, Tissue and Organ Culture: Basic Methods], ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-6; Weissinger et al. (1988) Ann Rev Genet 22:421-77; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen (1991) In vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al. (1998) Theor Appl Genet [Theoretical and Applied Genetics] 96:319-24 (soybean); Datta et al., (1990) Biotechnology [Biotechnology] 8:736-40 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci.USA 85:4305-9 (maize); Klein et al. (1988) Biotechnology 6:559-63 (maize); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol 91:440-4 (maize); Fromm et al. (1990) Biotechnology 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al., (1985) The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-505 (electroporation); Li et al. (1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al. (1996) Nat Biotechnol 14:745-50 (maize transformed by Agrobacterium tumefaciens).

[0308] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting cells or organisms with viruses or viral nucleic acids. Typically, such methods involve incorporating polynucleotides into viral DNA or RNA molecules. In some instances, the target polypeptide can be initially synthesized as a part of a viral polyprotein, and then the viral polyprotein is processed in vivo or in vitro by proteolytic processing to produce a desired recombinant protein. It is known to express polynucleotides in plants and to express proteins encoded therein (involving viral DNA or RNA molecules), see, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.

[0309] A variety of transient transformation methods can be used to provide or introduce polynucleotides or recombinant DNA constructs into prokaryotic and eukaryotic cells or organisms. Such transient transformation methods include, but are not limited to, introducing polynucleotide constructs directly into plants.

[0310] Nucleic acids and proteins can be provided to cells by any method, including methods using molecules to promote the uptake of any or all components (e.g., cell penetrating peptides and nanocarriers) of the directed Cas system (proteins and / or nucleic acids). See also US20110035836 published on February 10, 2011 and EP 2821486 A1 published on January 7, 2015.

[0311] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts may be used, including plastid transformation methods, and methods for introducing polynucleotides into tissues from seedlings or mature seeds.

[0312] "Stable transformation" is intended to mean that a nucleotide construct introduced into an organism is integrated into the genome of the organism and can be inherited by its progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into an organism and is not integrated into the genome of the organism, or that a polypeptide is introduced into an organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0313] A variety of methods can be used to identify those cells with altered genomes at or near the target site without using a screenable marker phenotype. Such methods can be considered to be direct analysis of the target sequence to detect any changes in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blotting, and any combination thereof.

[0314] Cells and plants

[0315] The polynucleotides and polypeptides disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. Any plant (including monocots and dicots and plant elements) can be used with the compositions and methods described herein.

[0316] Examples of monocots that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), sorghum (Panicum miliaceum), foxtail millet (Setaria italica), foxtail millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum vulgare), aestivum, einkorn wheat (Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), bananas (Musa spp.), palms, ornamentals, turf grasses, and other grasses.

[0317] Examples of dicots that can be used include, but are not limited to, soybean (Glycine max), Brassica species (such as, but not limited to, rapeseed or canola) (Brassica napus and B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum), and the like.

[0318] Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), bananas (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), kiwi fruit (Olea europaea), walnut ... europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals and conifers.

[0319] Vegetables that can be used include tomatoes (Tomato), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums.

[0320] Conifers that can be used include pines, such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs, such as silver fir (Abies vancouverii); amabilis) and balsam fir (Abies balsamea); and cedars, such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis).

[0321] In certain embodiments of the present disclosure, fertile plants are plants that produce live male and female gametes and are self-fertile. Such self-fertile plants can produce progeny plants without the contribution of gametes from any other plant and the genetic material contained therein. Other embodiments of the present disclosure may involve the use of non-self-fertile plants because the plant does not produce viable or otherwise fertile male or female gametes or both.

[0322] The present disclosure can be used for breeding of plants comprising one or more introduced traits or edited genomes.

[0323] Characteristics

[0324] In some aspects, the subject disclosure relates to the identification and detection of coding sequences comprising agronomic traits. In other aspects, the economically important traits are selected from the group consisting of: herbicide tolerance, disease resistance, insect or pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, increased oil, enhanced nutritional content, increased growth rate, enhanced stress tolerance, preferred maturity, enhanced organoleptic properties, altered morphological characteristics and sterility.

[0325] Anti-insect

[0326] Various insect resistance genes can include coding sequences. Exemplary insect resistance coding sequences are known in the art. As examples of insect resistance coding sequences that can be operably linked to the regulatory elements disclosed herein, the following traits are provided. Exemplary lepidopteran resistance coding sequences are provided including: cry1A; cry1A.105; cry1Ab; cry1Ab (truncated); cry1Ab-Ac (fusion protein); cry1Ac (as sales); cry1C; cry1F (as (sold); cry1Fa2; cry2Ab2; cry2Ae; cry9C; mocry1F; pinII (proteinase inhibitor protein); vip3A(a); and vip3Aa20. Exemplary coding sequences for anti-coleopteran insects include: cry34Ab1 (as sales); cry35Ab1 (as dvsnf7; and mcry3A. Exemplary coding sequences for resistance to multiple insects include ecry31.Ab. The above list of insect resistance genes is not meant to be limiting. The present disclosure encompasses any insect resistance gene.

[0327] Herbicide tolerance

[0328] Various herbicide tolerance genes may include coding sequences. Exemplary herbicide tolerance coding sequences are known in the art. As embodiments of herbicide tolerance coding sequences that may be operably linked to the regulatory elements disclosed in the present subject, the following traits are provided. Glyphosate herbicides contain a mode of action by inhibiting the EPSPS enzyme (5-enolpyruvyl shikimate-3-phosphate synthase). The enzyme is involved in the biosynthesis of aromatic amino acids that are essential for the growth and development of plants. A variety of enzymatic mechanisms known in the art can be used to inhibit the enzyme. Genes encoding such enzymes may be operably linked to the genetic regulatory elements disclosed in the present subject. In an embodiment, selectable marker genes include, but are not limited to, genes encoding glyphosate resistance genes, including: mutated EPSPS genes, such as 2mEPSPS genes, cp4 EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosate degradation genes, such as glyphosate acetyltransferase genes (gat) and glyphosate oxidase genes (gox). These traits are currently used as Gly-Tol TM , GT and Roundup sales. Resistance genes for glufosinate and / or bialaphos compounds include dsm-2, bar and pat genes. The bar and pat traits are currently used as Also included are tolerance genes that provide resistance to 2,4-D, such as the aad-1 gene (note that the aad-1 gene has further activity against aryloxyphenoxypropionate herbicides) and the aad-12 gene (note that the aad-12 gene has further activity against acetoxyacetate to synthesize auxins). These traits are Crop Protection Technology. Resistance genes to ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) are known in the art. These resistance genes are most often caused by point mutations to the ALS encoding gene sequence. Other ALS inhibitor resistance genes include the hra gene, csrl-2 gene, Sr-HrA gene, and surB gene. Some of these traits are marketed under the trade name Sale. Herbicides that inhibit HPPD include pyrazolones, such as benzylpyrazone, pyraclostrobin and benzylpyraclostrobin; triketides, such as mesotrione, sulfotrione, cyclosulfone, benzobicyclonone; and diketonitriles, such as isoxaflutole. Known traits can tolerate these exemplary HPPD herbicides. Examples of HPPD inhibitors include the hppdPF_W336 gene (for resistance to isoxaflutole) and the avhppd-03 gene (for resistance to mesotrione). Examples of oxini herbicide tolerance traits include the bxn gene, which has been shown to be resistant to the herbicide / antibiotic bromoxynil. Resistance genes for dicamba include dicamba monooxygenase genes (dmo), as disclosed in International PCT Publication No. WO2008 / 105890. Resistance genes for PPO or PROTOX inhibitor herbicides (e.g., acifluorfen, fluazifop-butyl, fluazifop-butyl, pentothiopyralid, mesotrione, isopropylpyraclostrobin, pyraclostrobin, benitobar-butyl, azofopyralid, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, oxazolidinone, oxazolidinone, fluazifop-butyl, fluazifop-butyl, oxazolidinone, oxyfluorfen, lactofen, fomesulfuron, fluazifop-butyl, and sulfentrazone) are known in the art.Exemplary genes that confer resistance to PPO include overexpression of wild-type Arabidopsis PPO enzyme (Lermontova I and Grimm B, (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol 122:75–83.), B. subtilis PPO gene (Li, X. and Nicholl D. 2005. Development of PPO inhibitor-resistant cultures and crops. Pest Manag. Sci. 61:277-285 and Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim MK, Kuk YI, Han SU and Guh JO, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. [By expressing the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants to generate resistance to the diphenyl ether herbicide oxyfluorfen-butyl] Biosci Biotechnol Biochem 62:558–560). Resistance genes for pyridyloxy or phenoxypropionic acid and cyclohexanone include genes encoding ACCase inhibitors (e.g., Acc1-S1, Acc1-S2, and Acc1-S3). Exemplary genes that confer resistance to cyclohexanedione and / or aryloxyphenoxypropionic acid include haloxyfop-butyl, diclofenac, fenoxaprop-butyl acid, haloxyfop-butyl, and quizalofop-butyl. Finally, herbicides that can inhibit photosynthesis include triazines or benzonitrile, with tolerance provided by the psbA gene (tolerance to triazines), the ls+ gene (tolerance to triazines), and the nitrilase gene (tolerance to benzonitrile). The above list of herbicide tolerance genes is not meant to be limiting. The present disclosure encompasses any herbicide tolerance gene.

[0329] Agronomic traits

[0330] Various agronomic traits genes can include coding sequences. Exemplary agronomic traits coding sequences are known in the art. As embodiments of agronomic traits coding sequences that can be operably connected to the regulatory elements disclosed in the present subject, the following traits are provided. The delayed fruit softening provided by the pg gene inhibits the production of polygalacturonase that causes the decomposition of pectin molecules in the cell wall, thereby causing delayed softening of the fruit. In addition, the delayed acc gene fruit ripening / senescence inhibits the normal expression of the natural acc synthase gene, resulting in reduced ethylene production and delayed fruit ripening. The accd gene metabolizes the precursor of the fruit ripening hormone ethylene, resulting in delayed fruit ripening. Alternatively, the sam-k gene causes delayed ripening by reducing S-adenosylmethionine (SAM), a substrate for ethylene production. The drought stress tolerance phenotype provided by the cspB gene maintains normal cell function under water stress conditions by maintaining RNA stability and translation. Another example includes the EcBetA gene, which catalyzes the production of the osmoprotectant compound glycine betaine, conferring tolerance to water stress. In addition, the RmBetA gene catalyzes the production of the osmoprotectant compound glycine betaine, conferring tolerance to water stress. The bbx32 gene provides photosynthesis and yield increase, expressing a protein that interacts with one or more endogenous transcription factors to regulate the day / night physiological processes of the plant. Ethanol production can be increased by expressing the amy797E gene encoding a thermostable alpha-amylase, which can enhance the production of bioethanol by increasing the thermostability of the amylase used to degrade starch. Finally, a modified amino acid composition can be produced by the expression of the cordapA gene encoding a dihydropicolinate synthase, which increases the production of the amino acid lysine. The list of agronomic trait coding sequences is not meant to be limiting. The present disclosure encompasses any agronomic trait coding sequence.

[0331] DNA Binding Proteins

[0332] Various DNA binding transgenes / heterologous coding sequence genes / heterologous coding sequences can include coding sequences. Exemplary DNA binding protein coding sequences are known in the art. As embodiments of DNA binding protein coding sequences that can be operably connected to the regulatory elements disclosed in this theme, the following types of DNA binding proteins may include: zinc fingers, TALEN, CRISPR and meganucleases. The list of DNA binding protein coding sequences is not intended to be restrictive. The present disclosure encompasses any DNA binding protein coding sequence.

[0333] Small RNA

[0334] Various small RNA sequences can include coding sequences. Exemplary small RNA traits are known in the art. As embodiments of small RNA coding sequences that can be operably connected to the regulatory elements disclosed in the present theme, the following traits are provided. For example, by silencing the expression of the ACO gene encoding the ethylene-forming enzyme, the production of ethylene is suppressed, and the delayed fruit ripening / senescence of the anti-efe small RNA delays ripening. By inhibiting endogenous S-adenosyl-L-methionine, the lignin production of the ccomt small RNA is changed, thereby reducing the content of guanidino (G) lignin: trans-caffeoyloxy CoA 3-O-methyltransferase (CCOMT gene). In addition, the tolerance to black spot bruises in Solanum verrucosum can be reduced by the Ppo5 small RNA, which triggers the degradation of the Ppo5 transcript, thereby preventing the development of black spot bruises. Also included is the dvsnf7 small RNA, whose dsRNA includes a 240bp fragment of the Snf7 gene of the Western Corn Rootworm, which can inhibit the Western Corn Rootworm. Modified starch / carbohydrates can be produced by small RNAs, such as pPhL small RNA (degrades PhL transcripts to limit the formation of reducing sugars by starch degradation) and pR1 small RNA (degrades R1 transcripts to limit the formation of reducing sugars by starch degradation). In addition, benefits also include reduced acrylamide content caused by asn1 small RNA, which triggers Asn1 degradation to impair asparagine formation and reduce polyacrylamide content. Finally, the non-browning phenotype of pgas ppo suppressing small RNA leads to the suppression of PPO to produce apples with a non-browning phenotype. The above list of small RNAs is not meant to be limiting. The present disclosure encompasses any small RNA encoding sequence.

[0335] Selectable marker

[0336] Various selectable markers (also described as reporter genes) can include coding sequences. There are many methods for confirming the expression of selectable markers in transformed plants, including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, Northern blotting, immunological methods for detecting proteins expressed from vectors. However, the reporter genes observed when visually observing proteins usually produce colored products when expressed. Exemplary reporter genes are known in the art and encode β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP, Phi-YFP), red fluorescent protein (DsRFP, RFP, etc.), β-galactosidase, etc. (see Sambrook et al., Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 3rd edition, Cold Spring Harbor Press, New York, 2001, the contents of which are incorporated herein by reference in their entirety).

[0337] Transformed cells or tissues are selected using selectable marker genes. Selectable marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO), spectinomycin / streptomycin resistance (AAD) and hygromycin phosphotransferase (HPT or HGR), and genes conferring resistance to herbicidal compounds. Herbicide resistance genes usually encode modified target proteins that are insensitive to herbicides, or encode enzymes that can degrade herbicides in plants or detoxify them before they work. For example, resistance to glyphosate has been obtained by using genes encoding mutant target enzymes 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS). The genes and mutants of EPSPS are well known and are further described below. Resistance to glufosinate, bromoxynil and 2,4-dichlorophenoxyacetic acid (2,4-D) has been obtained by using bacterial genes encoding PAT or DSM-2, nitrilase, AAD-1 or AAD-12 (these are examples of proteins that detoxify their corresponding herbicides).

[0338] In an embodiment, herbicides can inhibit growing points or meristems, including imidazolinone or sulfonylurea, and acetohydroxy acid synthase (AHAS) and acetolactate synthase (ALS) resistance / tolerance genes for these herbicides are well known. Glyphosate resistance genes include mutant 5-enolpyruvate shikimate-3-phosphate synthase (EPSPs) and dgt-28 genes (by introducing recombinant nucleic acids and / or carrying out multiple in vivo mutagenesis to natural EPSPs genes), aroA genes, and glyphosate acetyltransferase (GAT) genes. Other phosphono-resistance genes include bar and pat genes from streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes, as well as pyridyloxy or phenoxy propionic acid and cyclohexanone (genes encoding ACCase inhibitors). Exemplary genes that confer resistance to cyclohexanedione and / or aryloxyphenoxypropionic acid (including haloxyfop-butyl, diclofop-butyl, fenoxaprop-butyl acid, haloxyfop-butyl and quizalofop-butyl) include genes for acetyl-CoA carboxylase (ACCase); Acc1-S1, Acc1-S2 and Acc1-S3. In embodiments, herbicides may inhibit photosynthesis, including triazines (psbA and 1s+ genes) or benzonitrile (nitrilase gene). In addition, such selectable markers may include positive selection markers, such as phosphomannose isomerase (PMI) enzymes.

[0339] In embodiments, selectable marker genes include, but are not limited to, genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydropicolinate synthase; tryptophan decarboxylase; dihydropicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionate dehalogenase; acetohydroxyacid synthetase; 5-enolpyruvate-shikimate-phosphate synthase (aroA); halogenated aryl nitrile hydrolase; acetyl-CoA carboxylase; dihydropterin synthase (sul I); and 32kD photosystem II polypeptide (psbA). One embodiment also includes a selectable marker gene encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin. The above list of selectable marker genes is not meant to be limiting.The present disclosure encompasses any reporter gene or selectable marker gene.

[0340] In certain embodiments, synthetic coding sequence is with optimal expression in plant.For example, in an embodiment, the coding sequence of gene has been modified by codon optimization to enhance expression in plant.Insecticide resistance transgenic, herbicide tolerance transgenic, nitrogen use efficiency transgenic, water use efficiency transgenic, nutritional quality transgenic, DNA combined transgenic or selectable marker transgenic / heterologous coding sequence can be optimized to express in specific plant species, or alternatively above-mentioned transgenic / heterologous coding sequence can be modified to optimally express in dicotyledonous or monocotyledonous plants.Plant preference codon can be determined from the codon with the highest frequency in the protein expressed in maximum amount in specific purpose plant species.In an embodiment, coding sequence, gene, heterologous coding sequence or transgenic / heterologous coding sequence are designed to express with higher level in plant, thereby cause higher transformation efficiency.The method of plant gene optimization is well known. Guidance on the optimization and generation of synthetic DNA sequences can be found, for example, in WO 2013016546, WO 2011146524, WO 1997013402, US Pat. No. 6166302, and US Pat. No. 5380831 (incorporated herein by reference).

[0341] Molecular confirmation and detection

[0342] Methods for confirming the presence of polynucleotide insertions in plant genomes are known in the art. For example, polynucleotide insertions in plant genomes can be detected, for example, by polymerase chain reaction (PCR). PCR detection is performed by using two oligonucleotide primers flanking the polymorphic segment of the polymorphism, followed by DNA amplification. This step includes heat denaturation of the DNA, followed by annealing the primers to their complementary sequences at low temperatures, and repeated cycles of extending the annealed primers with a DNA polymerase. Size separation of DNA fragments on agarose or polyacrylamide gels after amplification is an essential part of the method. Such selection and screening methods are well known to those skilled in the art. Molecular confirmation methods that can be used to identify transgenic plants are known to those skilled in the art. Several exemplary methods are further described below.

[0343] Molecular beacons for use in sequence detection have been described. In short, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA. The unique structure of the FRET probe causes it to contain a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one primer in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of one or more FRET probes to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. The fluorescent signal indicates the presence of the flanking genomic / transgenic insert sequence due to successful amplification and hybridization. Such a molecular beacon assay for detecting an amplification reaction is an embodiment of the subject disclosure.

[0344] Hydrolysis probe assay, or (Life Technologies, Foster City, CA), is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed with one oligonucleotide in the transgene and one oligonucleotide in the flanking genomic sequence for event-specific detection. The FRET probe and PCR primers (one primer in the insert DNA sequence and one primer in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. The fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization. Such a hydrolysis probe assay for detecting an amplification reaction is an embodiment of the subject disclosure.

[0345] The assay is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a genomic DNA sample containing a polynucleotide that has integrated a gene expression cassette is assayed using a polymerase chain reaction (PCR)-based assay (known as The method used in the practice of the subject disclosure is The assay can utilize a PCR assay mixture. The primers used in the PCR assay mixture may include at least one forward primer and at least one reverse primer. The forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide, and the reverse primer contains a sequence corresponding to a specific region of the genomic sequence. In addition, the primers used in the PCR assay mixture may include at least one forward primer and at least one reverse primer. For example, The PCR assay mixture may use two forward primers corresponding to two different alleles and one reverse primer. One of the forward primers contains a sequence corresponding to a specific region of the endogenous genomic sequence. The second forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide. The reverse primer contains a sequence corresponding to a specific region of the genomic sequence. Such a mixture used to detect the amplification reaction Assays are one embodiment of the subject disclosure.

[0346] In some embodiments, the fluorescent signal or fluorescent dye is selected from the group consisting of: HEX fluorescent dye, FAM fluorescent dye, JOE fluorescent dye, TET fluorescent dye, Cy3 fluorescent dye, Cy 3.5 fluorescent dye, Cy 5 fluorescent dye, Cy 5.5 fluorescent dye, Cy 7 fluorescent dye, and ROX fluorescent dye.

[0347] In other embodiments, the amplification reaction is performed using a suitable second fluorescent DNA dye that can stain cellular DNA in a concentration range detectable by flow cytometry and has a fluorescence emission spectrum that can be detected by a real-time thermal cycler. It should be understood by those of ordinary skill in the art that other nucleic acid dyes are known and are being identified. Any suitable nucleic acid dye with a suitable excitation and emission spectrum can be used, such as YO- SYTOX SYBR and

[0348] In another embodiment, next generation sequencing (NGS) can be used for detection. As described in Brautigma et al., 2010, DNA sequence analysis can be used to determine the nucleotide sequence of the isolated and amplified fragments. The amplified fragments can be separated and subcloned into a vector and sequenced using a chain terminator method (also known as Sanger sequencing) or dye terminator sequencing. In addition, the amplicon can be sequenced using next generation sequencing. NGS technology does not require a subcloning step and can complete multiple sequencing reads in a single reaction. Three NGS platforms are commercially available, the genome sequencer FLX from 454 Life Sciences / Roche, and the FLX 100 from Roche. TM , Illumina Genome Analyser from Solexa TM and Applied Biosystems’ SOLiD TM (Abbreviation: "sequencing by oligonucleotide ligation and detection"). In addition, two single-molecule sequencing methods are currently under development. These include TMTrue Single Molecule Sequencing (tSMS) from , and Single MoleculeReal Time from Pacific Biosciences TM Sequencing (SMRT).

[0349] The FLX genome sequencer sold by 454 Life Sciences / Roche TM It is a long-read NGS that uses emulsion PCR and pyrosequencing to generate sequencing reads. DNA fragments of 300-800 bp or libraries containing 3-20 kb can be used. These reactions can produce more than one million reads of approximately 250 to 400 bases per run, with a total output of 250 to 400 megabases. Compared with other NGS technologies, this technology can produce the longest reads, but the total sequence output per run is lower.

[0350] Solexa TM Illumina Genome Analyser for sale TM It is a short-read NGS that uses fluorescent dye-labeled reversible terminator nucleotides by synthesis method and based on solid phase bridge PCR sequencing. The construction of paired-end sequencing libraries containing up to 10kb DNA fragments can be used. The reaction produces more than 100 million short reads with a length of 35-76 bases. Each run can generate 3-6 billion bases of data.

[0351] Applied Biosystems TM The marketed sequencing oligonucleotide ligation and detection (SOLiD) system is a short-read technology. This NGS technology uses fragments of double-stranded DNA up to 10 kb. The system performs sequencing by ligating dye-labeled oligonucleotide primers and emulsion PCR to generate 1 billion short reads with a total sequence output of up to 30 Gigabits per run.

[0352] Helicos Bioscience TM tSMS and Pacific Biosciences TM SMRT takes a different approach, using a single DNA molecule to perform the sequence reaction. TM The system can generate up to 800 million short reads, totaling 21 billion bases per run. These reactions are completed using virtual terminator nucleotides labeled with fluorescent dyes, which are known as "synthesis sequencing" methods.

[0353] Pacific Biosciences TMThe SMRT Next Generation Sequencing System is marketed using real-time sequencing by synthesis. This technology can generate read lengths up to 1,000 bp, as it is not limited by reversible terminators. This technology can generate a raw read throughput equivalent to double the coverage of the diploid human genome per day.

[0354] Although the present invention has been clearly shown and described with reference to preferred embodiments and various alternative embodiments, it will be appreciated by those skilled in the art that various changes may be made to it in form and detail without departing from the spirit and scope of the present invention. For example, although the following specific examples may set forth the methods and embodiments described herein using specific plants, the principles in these examples may be applied to any plant. Therefore, it should be understood that the scope of the present invention is encompassed by the embodiments of the present invention described herein and in the specification, rather than by the specific examples exemplified below. For all purposes, all cited patents and publications mentioned in this application are incorporated herein by reference in their entirety, to the extent that they are each individually and specifically incorporated by reference.

[0355] Examples

[0356] The following are examples of specific embodiments of some aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. With regard to the numbers used (e.g., amounts, temperatures, etc.), efforts have been made to ensure their accuracy, but some experimental errors and deviations should still be allowed.

[0357] Example 1: Genome editing tools for DNA target site cleavage and promoter activation

[0358] In this example, methods are described for producing genome editing tools that are capable of cleaving a genomic DNA target site and activating a promoter inserted at the cleavage site in a cell, particularly a plant cell.

[0359] In one approach, a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease (although other RNA-guided nucleases such as, but not limited to, transposon-associated protein B (TnpB), Fanzor, or HERMES may also be used (Karvelis et al. (2021) Nature. [Nature] 599:692-696, Saito et al. (2023) Nature. [Nature] available at doi.org / 10.1038 / s41586-023-06356-2, or Jiang et al. (2023) bioRxiv. [Biological preprint] doi: https: / / doi.org / 10.1101 / 2023.06.13.544871)) is engineered to have dual functionality capable of either DNA target cleavage or DNA target transcriptional activation. First, the maize codon table was used to encode sequences of Cas endonucleases (such as, but not limited to, Cas9 from Streptococcus pyogenes (Spy) (SEQ ID NO: 1), Cas12f from Syntrophomonas palmitica (Spa) or its engineered variants (SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4), or Cas12f from Acidibacillus sulfuroxidans (Asu) or its engineered variants (SEQ ID NO: 5 and SEQ ID NO: 6)), transcription activation domains (TADs) (such as, but not limited to, the C-terminal acidic plant transcription activation domain from Arabidopsis thaliana cold binding factor 1 (CFB1) protein (SEQ ID NO: 7), nuclear localization sequences (NLSs) (such as, but not limited to, the single-component simian virus 40 nuclear localization signal (mNLS) (PKKKRKV) or the bipartite NLS of the VirD2 protein from Agrobacterium tumefaciens (bNLS) (SEQ ID NO: 8), and the like). NO:8)) was codon optimized, and its GC content was adjusted using standard techniques known in the art, and repetitive sequences and gene instability features, such as miniature inverted repeat transposable elements (MITEs) were removed. Next, the optimized sequences encoding TAD and NLS were attached to the ends of the optimized sequences encoding Cas endonucleases in a mNLS-Cas-bNLS-CBF1 configuration (although other configurations may also be used). The resulting gene was then synthesized (GenScript, USA) and cloned in a Gateway compatible plasmid DNA containing a polymerase II promoter and terminator by restriction enzyme digestion and connection. For expression in maize, this includes the maize ubiquitin (UBI) promoter and terminator.To further enhance expression, a 5' untranslated region (UTR) (such as, but not limited to, the maize UBI 5'UTR) and additional introns (such as UBI maize intron 1 and potato ST-LS1 intron 2) are used, although other introns may also work. Examples of maize cell optimized Cas DNA expression constructs are in. Figure 1 In the description.

[0360] Next, the small RNA (referred to herein as guide RNA (gRNA)) that guides the mNLS-Cas-bNLS-CBF1 chimeric protein described herein to the DNA target is engineered to specify target cleavage or promoter activation. To this end, the length of the variable targeting (VT) domain of the gRNA is adjusted, which guides mNLS-Cas-bNLS-CBF1 to its DNA target by base pairing with one strand of the DNA target site adjacent to a suitable protospacer sequence or target adjacent motif (PAM or TAM) (5'-TTC-3' for SpaCas12f1, 5'-TTN-3' for AsuCas12f1, and 5'-NGG-3' for SpyCas9). For DNA target cleavage, a VT length of 17-20nt was used, while for DNA target binding and promoter activation, a VT length of 10-15nt was used, similar to that described previously (Dahlman et al. (2015) Nat. Biotechnol. [Nature Biotechnology] 33: 1159-1161). Next, the 5' and 3' ends of the encoding gRNA sequence were operably linked to the U6 polymerase III promoter and terminator (TTTTTTTT). Then, the resulting U6 promoter-gRNA-U6 terminator expression cassette (GenScript, USA) was synthesized and cloned by restriction enzyme digestion and ligation into Gateway compatible plasmid DNA. In order to promote optimal transcription of gRNA from the U6 promoter, G nucleotides were added to the 5' end of the sequence to be transcribed. Ribozyme motifs (Gao et al. (2014) J. Integr Plant Biol. [Journal of Integrated Plant Biology] 56: 343-349), RNase P and Z cleavage sites (Xie et al. (2015) Proc. Natl. Acad. Sci. USA. [Proceedings of the National Academy of Sciences of the United States] 112: 3570-3575) and / or Csy4 (Cas6 or CasE) ribonuclease recognition sites (Tsai et al. (2014) Nat. Biotechnol. [Natural Biotechnology] 32: 569-576) can also be used to help the maturation of gRNA transcripts after transcription. In addition, the RNA processing provided by these strategies can also be used to express multiple gRNAs from a single polymerase II or III promoter (Gao et al. (2014), Xie et al. (2015) and Tsai et al. (2014)). Examples of Cas gRNA expression constructs optimized for maize are in Figure 2 In the description.

[0361] In another method, two orthogonal genome editing tools are used, one for cutting the DNA target site, and the other for activating the promoter. This can cover RNA-guided editing tools, those that do not require gRNA (such as but not limited to zinc finger nucleases (Urnov et al. (2010) Nature Reviews Genetics. [Genetics Natural Review] 11: 636-636), TALE nucleases (Becker et al. (2021) Gene and Genome Editing. [Gene and genome editing] 2: 100007) or meganucleases (Zekonyte et al. (2021) Nature Communications. [Natural Communications] 12: 3210)), or a combination of different tools. For this reason, the sequence encoding the genome editing enzyme is adjusted as described above for expression. Next, for the genome editing protein intended to activate the promoter, alanine or other suitable amino acids are substituted into one or more key residues required for DNA target cutting, or in the case of zinc fingers or TALE nucleases, the FokI nuclease domain is removed. This enables the genome editing protein to bind but not cut the DNA site. As described above, the sequences encoding the TAD and NLS were then ligated in-frame with the sequence encoding the nuclease death (d) protein. The sequence encoding the genome editing nuclease was fused in-frame only to the sequence encoding the NLS. The two chimeric genes were then synthesized (GenScript, USA) and cloned using restriction enzyme digestion and ligation into a Gateway-compatible plasmid DNA containing the promoter, 5'UTR, introns and terminator as described above. The gRNA was optimized for expression as described above, except that a gRNA with a length of only 17-20nt VT was used. An example expression cassette is shown in Figure 3 middle.

[0362] Example 2: DNA repair template for site-specific insertion

[0363] In this example, methods are described for generating DNA repair templates for site-specific insertion into a genomic DNA target in a cell, particularly a plant cell.

[0364] In one method, additional DNA sequences are appended to the 5' and 3' ends of the DNA sequence to be inserted (such as, but not limited to, a gene conferring a desired trait). This includes a minimal promoter, a suboptimal promoter, or a non-functional promoter (such as, but not limited to, a minimal 35S cauliflower mosaic virus promoter), a 5'UTR (such as, but not limited to, a 5'UTR from tobacco mosaic virus (TMV)), a selectable marker (such as, but not limited to, a sequence encoding neomycin phosphotransferase II (NPTII) (SEQ ID NO: 60), plant phosphomannose isomerase (PMI) (SEQ ID NO: 61), Wuschel2 (Wus2) (SEQ ID NO: 62), BabyBoom ​​(Bbm) (SEQ ID NO: 63), coral reef red fluorescent protein (DsRed) from a species of the genus Discosoma (Discosoma) (SEQ ID NO: 64), or a fusion between the maize optimized phosphinothricin acetyltransferase (moPAT) and DsRed (moPAT-DsRed) protein (SEQ ID NO: 65). NO:65)) and a terminator (such as, but not limited to, the T28 terminator from rice), optionally a DNA sequence homologous to one or both sides of the genomic DNA target cleavage site, and optionally one or more DNA cleavage sites. The resulting DNA repair template is then synthesized (GenScript, USA) and restriction enzymes are ligated into Gateway compatible plasmid DNA using methods known in the art. A schematic diagram of a DNA repair template for site-specific insertion into a genomic DNA target of a cell is shown in Figure 4 middle.

[0365] Example 3: Transformation of components for site-specific DNA insertion

[0366] In this example, methods are described for introducing one or more genome editing tool expression cassettes and DNA repair templates for site-specific insertion into genomic DNA targets in cells, particularly plant cells.

[0367] One or more genome editing tool expression cassettes and a DNA repair template are co-delivered on a single transfer DNA (T-DNA) into maize immature embryos using Agrobacterium as previously described (Lowe et al. (2018) In vitro cellular & developmental DNA) although other transformation methods may be used, such as but not limited to biolistics, Ensifer-based, nanoparticle-mediated, or protoplast-utilizing methods (Rathore et al. (2019) Transgenic Plants: Methods and Protocols. New York, NY: Springer New York, 37-48, Wang et al. (2019) Molecular Plant. 12, 1037-1040, Rhodes et al. (1988) Science. 240, 204-207, and Golovkin et al. (1993) Plant Science. 90, 41-52). biology-Plant. [In vitro cell and developmental biology-plant] 54, 240-252). Briefly, one or more optimized genome modification tool expression cassettes and a DNA repair template Gateway are cloned into a T-DNA, which optionally already contains genes encoding the morphogenic transcription factors Bbm and Wus2 operably linked to the Zm-PLTP and Axig1 promoters, respectively, if not used in the repair template. The resulting T-DNA is then placed into LBA4404, a thymine auxotrophic strain of Agrobacterium tumefaciens containing Vir9, a separate plasmid encoding the Bo542 virulence gene (US20170121722 A1 and WO 2017 / 078836). Then the resulting Agrobacterium strain is used to transform the immature maize embryo (about 2mm in size) of nine to ten days old, and the transformation is continued for 5min by being immersed in the 700A liquid medium (optical density at 550nm is 0.7) containing the strain, then taken out from the culture medium, and placed in a solid co-culture medium, in the dark at 21°C overnight. After T-DNA delivery, the embryo is transferred to the resting culture medium 13266R, and grows in the dark at 28°C for 5-7 days. Next, the coleoptile is removed, and the embryo is transferred to the selection culture medium for 11-16 days, and maintained in the dark at 28°C. Then they are moved to the mature culture medium, and hatched at 28°C for 14-25 days in the dark, and then hatched under illumination for 2-5 days. Next, they are transferred to the rooting culture medium, and hatched under 26-28°C illumination for 14-28 days, and the culture medium is updated as needed.To foster optimal conditions for activity of the genome editing tools, the transformed tissues were incubated at 28°C (standard for tissue culture) or at a range of temperatures below or above 28°C.

[0368] Example 4: Detection of site-specific DNA insertion in cells

[0369] In this example, methods for detecting site-specific DNA insertions in cells, particularly plant cells, are described.

[0370] In one approach, PCR was used to detect site-specific DNA insertions in plant cells as previously described (Peterson et al. (2021) Plant Biotechnol. J. 19:2000-2010). Briefly, primers were designed and synthesized (Integrated DNA Technologies, USA) to amplify each junction ( Figure 5 Next, T0 plants were sampled (V2 or V3 leaf punch) and DNA was extracted using the Synergy 2.0 Plant DNA Extraction Kit (Ops Diagnostics, USA). Next, T0 plants containing putative insertions were screened using quantitative PCR (qPCR). In this case, TaqMan probes (Integrated DNA Technologies, USA) were also designed, synthesized, and used with the aforementioned primers ( Figure 5 ). Next, plants with a positive qPCR signal were subjected to additional analysis relative to a negative control (untransformed wild-type genomic DNA). This included PCR amplification and fragment size analysis using agarose gel electrophoresis of each genomic and insertion junction. T0 plants that showed amplification products of the correct size at both junctions were also subjected to long PCR. To this end, forward and reverse primers outside the DNA insertion in the flanking genomic regions were used to amplify the entire DNA insertion, and the presence of a PCR product of the expected size was used to confirm the presence of the complete site-specific DNA insertion ( Figure 5 ).

[0371] Example 5: Selection for site-specific DNA insertion

[0372] In this example, methods are described for selecting DNA inserted at or near a genome editing tool DNA target cleavage site by activating a promoter.

[0373] In one approach, cells or plant parts containing site-specific DNA insertions are enriched by activating a promoter operably linked to a selectable marker. Figure 6)。To this end, a genome modification tool is used to cleave a DNA target site in the presence of a DNA repair template containing one or more genes of interest and a minimal, suboptimal, or non-functional promoter driving the expression of a selectable marker. Figure 6 )。Then, a genome modification tool that can bind but not cleave one or more DNA target sites is directed to genomic regions flanking the cleaved DNA target such that expression of the selectable marker is activated only in cells or plants containing a site-specific DNA insertion. Figure 6 )。Next, a selection agent (chemical or visual) is applied to enrich cells or plants containing a site-specific DNA insertion. Using this method, the orientation of the inserted DNA can also be controlled by the positioning of the promoter-activated target site. Figure 7 )。If located 5' of the cleavage site, DNA insertions will occur in the forward or sense orientation, and if located 3' of the cleavage site, they will insert in the reverse or antisense orientation. Figure 7 )。Different selectable markers can also be used simultaneously to achieve multiple DNA insertions. If desired, an inducible site-specific recombinase (Cre) can be used to excise the selectable marker after selection, as previously described for the removal of morphogenic genes (Wang et al. (2020) Frontiers in Plant Science. 11:1298). Alternatively, sequences homologous to the genomic DNA flanking the cleavage site can be placed between the selectable marker and the gene of interest. Figure 8 )。And then, upon cleavage by the targeted insertion sequence, intramolecular repair between directly repeated homologous sequences, as previously described for the reconstruction of the β-glucuronidase (GUS) gene (Orel et al. (2003) Plant J. 35:604–612), can be used to excise the selectable marker gene in a scarless manner. Figure 8 )。

[0374] Example 6: Efficient recovery of site-specific DNA insertions in plants

[0375] In this example, the method described herein is used to improve the recovery of targeted DNA insertions in plants.

[0376] Transformation was performed as described above using Agrobacterium and T0 maize plantlets expressing DsRed identified using the Xite fluorescent flashlight system (NIGHTSEA, USA). After sampling from the red fluorescent plants, genomic DNA was extracted and the presence of site-specific gene insertions was evaluated, as described in Example 4. The control utilized both mNLS-SpyCas9-bNLS and mNLS-SpyCas9-bNLS-CBF1, and only included one gRNA that could cut the genomic target site and excise the repair template. Promoter activation experiments used mNLS-SpyCas9-bNLS-CBF1, one gRNA that cuts the genomic target site and excises the repair template, and four, five or six promoters that activate the gRNA (pagRNA). The control DNA repair template is depicted in Figure 4 For activation experiments, use Figure 4 iv. Figure 4 iii and Figure 4 Four, five, and six pagRNAs for the DNA repair template combinations shown in ii.

[0377] like Fig. 9 As shown, 71%, 84% and 96% of T0 plantlets were observed to be PCR positive for junction 1 (a unique junction generated by inserting a minimal promoter and selectable marker gene into the SpyCas9 genomic cleavage site) when six, five and four pagRNAs were used, respectively. In the absence of activation, the frequency of plants positive for junction 1 was between 5% and 6% ( Fig. 9 ). Next, to assess the completeness of the targeted insertion, the frequency of plants containing both junctions 1 and 2 was determined. Fig. 9 As shown, approximately half of the plants positive for junction 1 in the activation experiment were also positive for insertion of junction 2, resulting in 33%, 45%, and 54% of plants with predicted complete target gene insertions. Overall, a 7- to 14-fold increase in the recovery of targeted gene insertions was observed relative to experiments not utilizing promoter activation.

[0378] Example 7: Use of site-specific DNA insertion to select for chromosomal DNA crossovers, inversions or relocations

[0379] In this example, methods for selecting for chromosomal DNA crossovers, inversions, or relocations at or near a genome modification tool DNA target cleavage site are described.

[0380] In one method, a minimal promoter, suboptimal promoter, or non-functional promoter operably linked to a selectable marker (SM) is first inserted into the border of a chromosomal region that is scheduled to undergo a crossover, inversion, or relocation using methods described herein. For a crossover, the 5' region of the minimal promoter, suboptimal promoter, or non-functional promoter (MSNP) is then targeted for cleavage by a genome modification tool ( Fig.10 In the case of inversion, the 5' region of the MSNP and the distal boundary (DB) of the target chromosome region are targeted for cleavage ( Fig.11 ). For relocation, the 5' region, DB and new chromosomal location of MSNP were targeted for cleavage ( Fig.12 Next, the desired DNA repair outcome is selected using genome modification tools designed to activate transcription of the selectable marker ( Figure 10-12 As described in Example 5, the selectable expression cassette can be excised if desired.

Claims

1. A method of editing the genome of a target cell to include a donor polynucleotide, the method comprising: (a) inserting a donor polynucleotide into a genomic target site, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence, such that the coding sequence is not expressed or is expressed at a low level; (b) binding a site-specific promoter activation complex to a genomic region upstream of the inserted donor polynucleotide, wherein the site-specific promoter activation complex functions to drive the non-functional promoter to express a protein from the coding sequence; (c) expressing the coding sequence at a higher level; (d) selecting target cells expressing the coding sequence; as well as (e) obtaining target cells expressing the coding sequence.

2. The method of claim 1, wherein the coding sequence comprises an agronomic trait, wherein the agronomic trait is selected from the group consisting of an insecticide resistance trait, a herbicide tolerance trait, a nitrogen use efficiency trait, a water use efficiency trait, a nutritional quality trait, a DNA binding trait, a small RNA trait, a selectable marker trait, or any combination thereof.

3. The method of claim 1, wherein the coding sequence comprises a selectable marker.

4. The method of claim 4, wherein the selectable marker is selected from the group consisting of nptll, pat, bar, dsm-2, ahas, gox, gat, gus, fluorescent protein, or any combination thereof.

5. The method of claim 1, wherein the coding sequence confers resistance to a herbicide or an antibiotic.

6. The method of claim 1, wherein the non-functional promoter comprises a minimal promoter.

7. The method of claim 6, wherein the minimal promoter comprises a TATA box, a CAAT box, a transcription start site, an RNA polymerase binding site, or any combination thereof.

8. The method of claim 1, wherein the non-functional promoter drives low levels of the coding sequence.

9. The method of claim 1, wherein the site-specific promoter activation complex comprises a site-specific binding protein operably linked to at least one activation domain.

10. The method of claim 9, wherein the site-specific binding protein is a CRIPSR protein, a zinc finger protein or a TALEN protein.

11. The method of claim 10, wherein the CRISPR protein is mutagenized to inactivate nuclease activity.

12. The method of claim 9, wherein the activation domain is RTA, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, VP64, VP16, VP160, GAL4, EDLL, ERF2, CBF1, ORCA2, DREB1A, LEAFY, or any combination thereof.

13. The method of claim 1, wherein the site-specific promoter activation complex binds in the vicinity of the non-functional promoter.

14. The method of claim 1, wherein the site-specific promoter activation complex binds within 1-10,000 bp of the non-functional promoter.

15. The method of claim 1, wherein the coding sequence is expressed at a higher level, the higher level being 1% to 100% higher than an initial low level of expression of the coding sequence.

16. The method of claim 1, wherein the expression of the coding sequence driven by the site-specific promoter activation complex is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold greater than the expression of the coding sequence driven only by the non-functional promoter.

17. The method of claim 1, wherein multiple copies of the site-specific promoter activation complex bind to a genomic region upstream of the inserted donor polynucleotide such that the site-specific promoter activation complex functions to drive the non-functional promoter.

18. The method of claim 1, wherein the cell is a plant cell.

19. The method of claim 18, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

20. A method for modifying the genome of a plant cell, the method comprising: (a) cleaving a genomic target site within the genome of the plant cell; (b) introducing a donor polynucleotide into the plant cell, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence, such that the coding sequence is not expressed or is expressed at a low level; (c) inserting the donor polynucleotide into the genome of the plant cell; (d) introducing a site-specific promoter activation complex into the plant cell; (e) binding the site-specific promoter activation complex to the genome of the plant cell, wherein the site-specific promoter activation complex binds upstream of the non-functional promoter, wherein the site-specific promoter activation complex acts to drive the non-functional promoter to express a protein from the coding sequence; (f) expressing the coding sequence at a higher level; (g) selecting said plant cell expressing said coding sequence; and (h) obtaining the plant cell expressing the coding sequence.

21. A method for modifying the genome of a plant cell, the method comprising: (a) introducing a donor polynucleotide into a first homologous chromosome in the genome of the plant cell, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence, such that the coding sequence is not expressed or is expressed at a low level; (b) cleaving the genomic DNA of the first homologous chromosome upstream of the donor polynucleotide with a site-specific nuclease; (c) cleaving the genomic DNA of the second homologous chromosome with a site-specific nuclease; (d) recombining the genomic DNA of the first chromosome with the genomic DNA of the second homologous chromosome; (e) generating a modified genome, wherein the second homologous chromosome is located upstream of the donor polynucleotide; (f) introducing a site-specific promoter activation complex into the plant cell; (g) binding the site-specific promoter activation complex to the second homologous chromosome, wherein the site-specific promoter activation complex binds upstream of the non-functional promoter, so that the site-specific promoter activation complex acts to drive the non-functional promoter to express a protein from the coding sequence; (h) expressing the coding sequence at a higher level; (i) selecting a plant cell that expresses the coding sequence and contains the donor polynucleotide recombined into the second homologous chromosome; and, (j) obtaining a plant cell expressing the coding sequence and containing the donor polynucleotide recombined into the second homologous chromosome.

22. A method for modifying the genome of a plant cell, the method comprising: (a) introducing a donor polynucleotide into a chromosome in the genome of the plant cell in a cis orientation, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence, such that the coding sequence is not expressed or is expressed at a low level; (b) introducing a site-specific promoter activation complex into the plant cell; (c) cleaving the genomic DNA of the first chromosome at sites upstream and downstream of the donor polynucleotide with a site-specific nuclease; (d) inserting the donor polynucleotide into a chromosome within the genome of the plant cell in a trans orientation; (e) binding the site-specific promoter activation complex to the second chromosome, wherein the site-specific promoter activation complex binds upstream of the non-functional promoter, wherein the site-specific promoter activation complex acts to drive the non-functional promoter to express a protein from the coding sequence; (f) expressing the coding sequence at a higher level; (g) selecting a plant cell that expresses the coding sequence and contains the donor polynucleotide, the donor polynucleotide being inserted into a chromosome within the genome of the plant cell in the trans orientation; and, (h) obtaining a plant cell containing the donor polynucleotide, wherein the donor polynucleotide is inserted into a chromosome in the genome of the plant cell in the trans orientation.

23. A method for modifying the genome of a plant cell, the method comprising: (a) introducing a donor polynucleotide into a first chromosome in the genome of the plant cell, wherein the donor polynucleotide comprises a gene expression cassette comprising a non-functional promoter operably linked to a coding sequence, such that the coding sequence is not expressed or is expressed at a low level; (b) cleaving the genomic DNA of the first chromosome upstream of the donor polynucleotide with at least one site-specific nuclease; (c) cleaving the genomic DNA of the second chromosome with at least one site-specific nuclease; (d) recombining the genomic DNA of the first chromosome with the genomic DNA of the second chromosome; (e) generating a modified genome, wherein the second chromosome is located upstream of the donor polynucleotide; (f) introducing a site-specific promoter activation complex into the plant cell; (g) binding the site-specific promoter activation complex to the second chromosome, wherein the site-specific promoter activation complex binds upstream of the non-functional promoter, wherein the site-specific promoter activation complex acts to drive the non-functional promoter to express a protein from the coding sequence; (h) expressing the coding sequence at a higher level; (i) selecting a plant cell that expresses the coding sequence and contains the donor polynucleotide recombined into the second chromosome; and, (j) obtaining a plant cell expressing the coding sequence and containing the donor polynucleotide recombined into the second chromosome.

Images