Two-step gene exchange

Abstract

Compositions and methods for excising and replacing endogenous polynucleotides, such as genes, using CRISPR-Cas systems are provided. In some aspects, the gene is flanked by specific nucleotides that are targets for homology-directed repair to insert replacement polynucleotides. Methods and compositions for replacing polynucleotides in genomes containing highly repetitive regions and for improving the phenotype of an organism are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 958,805, filed on January 9, 2020, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of plant molecular biology, and more particularly to compositions and methods for altering the genome of a cell. Background Art

[0004] Recombinant DNA technology makes it possible to insert DNA sequences at target genomic locations and / or modify specific endogenous chromosomal sequences. Site-specific integration techniques using site-specific recombination systems and other types of recombination techniques have been used to produce targeted insertions of target genes in various organisms. Genome editing technologies such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or homing meganucleases can be used to produce targeted genomic perturbations, but these systems tend to have low specificity and use designed nucleases that need to be redesigned for each target site, making them expensive and time-consuming to prepare.

[0005] A newer technology that exploits the adaptive immune system of archaea or bacteria, called CRISPR (clustered regularly interspaced short palindromic repeats ( C lustered R egularly I Interspaced S hort P alindromic R epeats)), which contain different domains of effector proteins that encompass multiple activities (DNA recognition, binding and optionally cleavage).

[0006] Direct targeting of DNA double-strand breaks (DSBs) has been shown to significantly increase the frequency and accuracy of genome editing. CRISPR-Cas technology has been successfully applied to genome editing in a variety of plant species, however, the success rates of different types of genome editing applications vary greatly. Replacement of a gene or any part of a gene or its promoter is difficult as it requires two events to occur - the deletion of the gene / gene fragment and the insertion of the new sequence in a very precise manner by homology-directed repair (HDR).

[0007] Despite the identification of some of these systems, there remains a need for methods and compositions for increasing the frequency and / or efficacy of replacing endogenous polynucleotides in the genome of a cell. Summary of the invention

[0008] Provided are methods and compositions for replacing a polynucleotide in a cell genome with another polynucleotide, such as replacing an allele responsible for disease susceptibility with an allele that provides disease tolerance. Also provided are methods and compositions for replacing polynucleotides in a genome containing highly repetitive regions and for improving the phenotype of an organism.

[0009] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0010] The present disclosure can be more fully understood from the following detailed description and accompanying drawings and sequence listing, which form a part of this application.

[0011] Figure 1 Depicted is a two-step method for replacing one (e.g., endogenous) polynucleotide sequence in a cell's genome with another (replacement) polynucleotide. WUS = Wuschel morphogen; BBM = Babyboom morphogen; Cas9 = representative Cas endonuclease; gRNA = guide RNA; NPTII = terminator; TS1 = target site 1; TS2 = target site 2.

[0012] Figure 2 Schematic representation of a portion of maize chromosome 8, which contains multiple northern leaf blight (NLB) disease susceptibility / resistance loci.

[0013] Figure 3 Schematic diagram of creating disease-resistant inbred corn lines.

[0014] Figure 4 A two-step gene exchange in maize replacing a disease susceptibility gene with a disease resistance gene at the NLB18 locus is shown to confer a disease resistance phenotype to maize plants.

[0015] Figure 5 Gene expression at the NLB18 locus in maize is shown for expression of four allelic exchange lines. Hom = homozygous with 2 copies of the NLB18 gene; null = segregant without NLB18-BC26B from the same transformation; TI = NLB18-BC26N introgressed into the same line; WT = wild type germplasm, without NLB18-BC26N. DETAILED DESCRIPTION

[0016] Disclosed herein are methods and compositions for two-step exchange of endogenous polynucleotides and replacement with heterologous replacement polynucleotides.The endogenous polynucleotide may be naturally occurring within the genome of the host organism, or may be heterologous and previously introduced.

[0017] 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.

[0018] As used herein, "nucleic acid" means polynucleotides, and includes a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Therefore, the terms "polynucleotides", "nucleic acid sequences", "nucleotide sequences" and "nucleic acid fragments" 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 as follows with their single-letter names: "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.

[0019] The term "genome" when applied to a prokaryotic or eukaryotic cell or cell of an organism encompasses not only the chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components of the cell (eg, mitochondria, or plastids).

[0020] "Open reading frame" is abbreviated as ORF.

[0021] The term "selectively hybridizes" includes reference to hybridization of a nucleic acid sequence to a specific nucleic acid target sequence under stringent hybridization conditions to a detectably greater extent (e.g., at least 2 times background) than hybridization to non-target nucleic acid sequences and substantially to the exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) to each other.

[0022] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize with its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different situations. By controlling the stringency of hybridization conditions and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, optionally less than 500 nucleotides in length. Typically, stringent conditions will be conditions where the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other one or more salts) at pH 7.0 to 8.3, and is at least about 30°C for short probes (e.g., 10 to 50 nucleotides), and is at least about 60°C for long probes (e.g., more than 50 nucleotides). Adding a destabilizing agent such as formamide can also achieve stringent conditions. Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1X to 2X SSC (20X SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary medium stringency conditions include hybridization at 37° C. in 40% to 45% formamide, 1 M NaCl, 1% SDS, and washing in 0.5X to 1X SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization at 37° C. in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1X SSC at 60° C. to 65° C.

[0023] "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. The homologous region can have any length sufficient to promote homologous recombination at the target site of cleavage. For example, the length of the 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, 5-1500, 5-1600, 5-1700, 5-1800, 5-2000 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases so that the homologous region has enough homology to carry out homologous recombination with the corresponding genomic region. "Sufficient homology" means that two polynucleotide sequences have enough structural similarity 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.

[0024] As used herein, a "genomic region" is a segment of a chromosome in the genome of a cell present on either side of a target site, or alternatively, also comprising a portion of a target site. A genomic region may comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000 00, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases so that the genomic region has sufficient homology to undergo homologous recombination with the corresponding homologous region.

[0025] As used herein, "homologous recombination (HR)" includes the exchange of DNA fragments between two DNA molecules at homologous sites. The frequency of homologous recombination is affected by multiple factors. Different organisms vary with respect to the amount of homologous recombination and the relative proportion of homologous and non-homologous recombination. Generally, 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 only 25-50bp of homology has been observed. See, for example, 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.

[0026] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0027] "Percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein when comparing the optimal alignment of two sequences with a reference sequence (which does not include additions or deletions), the portion of the polynucleotide or polypeptide sequence in the 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 the result is then 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 percentage from 50% to 100%. These identities can be determined using any program described herein.

[0028] Sequence alignments 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 MegAlign® of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). TM Program. In the context of this application, it will be understood that, where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" will mean any set of values ​​or parameters that are initially loaded into the software when it is first initialized.

[0029] 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 MegAlign of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). TMProgram. For multiple alignments, the default values ​​correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignments and percentage identity calculations 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 DIAGONALS SAVED = 4. After aligning sequences using the Clustal V program, it is possible to obtain "percent identity" by viewing the "sequence distance" 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 found in MegAlign of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). TMv6.1 program. 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 the "Percent Identity" by viewing the "Sequence Distance" 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 89: 10915). GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol 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 a search algorithm for finding the region of similarity between biological sequences provided by the National Center for Biotechnology Information (NCBI). The program compares nucleotide or protein sequences with sequence databases, and calculates the statistical significance of the match to identify sequences with enough similarity to the query sequence, so that similarity is not predicted to have occurred randomly. The sequences identified by BLAST report and their local comparison with the query sequence. 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, wherein such polypeptides 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 percentage from 50% to 100%.In fact, any amino acid identity from 50% to 100% will be useful in describing the present disclosure, such as 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%.

[0030] Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described using the terms "homology", "homologous", "substantially identical", "substantially similar", and "substantially corresponding", which are used interchangeably herein. These refer to polypeptide 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 a 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 a nucleic acid fragment. Substantially similar nucleic acid sequences encompassed can be defined by the ability of these nucleic acid sequences to hybridize with the sequences exemplified herein, or to hybridize with any portion of a nucleotide sequence disclosed herein and functionally equivalent to any nucleic acid sequence disclosed herein (under moderate stringency conditions, such as 0.5X SSC, 0.1% SDS, 60°C). Stringent conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms) to highly similar fragments (such as genes that replicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions.

[0031] 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.

[0032] An "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, or a biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide does not contain sequences that naturally flank the polynucleotide (i.e., sequences at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived (optimally protein coding sequences). For example, in various embodiments, the isolated polynucleotide may comprise a nucleotide sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Isolated polynucleotides may be purified from cells in which they naturally occur. Conventional nucleic acid purification methods known to the skilled artisan may be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0033] The term "fragment" refers to a continuous set of nucleotides or amino acids. 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 nucleotides. 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 amino acids. A fragment may or may not show the function of a sequence that shares a certain percentage of identity over the length of the fragment.

[0034] The terms "functionally equivalent fragments" and "functionally equivalent fragments" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated 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 an active protein, the fragment retains the ability to alter gene expression or produce a certain phenotype. For example, fragments can be used to design genes to produce a desired phenotype in a modified plant. A gene can be designed for use in inhibition, whether or not the gene encodes an active enzyme, by connecting its nucleic acid fragment in a sense or antisense orientation relative to a plant promoter sequence.

[0035] "Gene" includes a nucleic acid fragment that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene found in its natural endogenous location with its own regulatory sequences.

[0036] The term "endogenous" refers to 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.

[0037] "Allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all 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.

[0038] "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 or downstream (3' non-coding sequence) of a coding sequence, and which 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.

[0039] A "mutant gene" is a gene that has been altered by human intervention. Such a "mutant gene" has a sequence that differs from the sequence of the corresponding non-mutant 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.

[0040] As used herein, the term "targeted mutation" refers to a mutation in a gene (referred to as a target gene), including a natural gene, generated by altering a target sequence within a target gene using any method known to those skilled in the art, including a method involving a guided Cas endonuclease system as disclosed herein.

[0041] The terms "knockout", "gene knock-out" and "genetic knock-out" are used interchangeably herein. Knockout means that a DNA sequence of a cell has been partially or completely inactivated by targeting with a Cas protein; for example, such a DNA sequence may have encoded an amino acid sequence before knockout, or may have had a regulatory function (e.g., a promoter).

[0042] 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 Cas protein (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.

[0043] "Domain" means a continuous stretch of nucleotides (which may be RNA, DNA, and / or RNA-DNA combination sequences) or amino acids.

[0044] The term "conserved domain" or "motif" refers to a group of polynucleotides or amino acids that are conserved at specific positions along aligned sequences of evolutionarily related proteins. Although amino acids at other positions may 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 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.

[0045] 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.

[0046] An "optimized" polynucleotide is a sequence that has been optimized for improved expression in a specific heterologous host cell.

[0047] 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, for example, a Cas endonuclease as disclosed herein, using one or more plant preferred codons to improve expression. For a discussion of host preferred codon usage, see, e.g., Campbell and Gowri, (1990) Plant Physiol. 92: 1-11.

[0048] Promoter is the 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 element and a more 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 of the promoter or tissue specificity. Promoters can all be derived from natural genes, or are 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, due to the fact that the exact boundaries of the regulatory sequence are not yet fully defined in most cases, some DNA fragments of variations may have the same promoter activity.

[0049] 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 endogenous or exogenous stimuli, such as by chemical compounds (chemical inducers), or to environmental, hormone, chemical, and / or developmental signals. Inducible or regulated promoters include, for example, promoters that are induced or regulated 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).

[0050] "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 mRNA upstream of the translation initiation sequence. The translation leader sequence can affect the processing of the mRNA by the primary transcript, 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).

[0051] "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 capable of affecting 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 various 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1: 671-680.

[0052] "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. "Justice" RNA refers to an RNA transcript that contains 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 specific gene transcript, i.e., a 5' non-coding sequence, a 3' non-coding sequence, an intron or a coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but still has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts and are intended to define the antisense RNA of the messenger.

[0053] The term "genome" refers to the entire complementary sequence of genetic material (genes and non-coding sequences) present in every cell of an organism or virus or organelle; and / or the complete set of chromosomes inherited as a (haploid) unit from one parent.

[0054] 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 (i.e., the coding sequence is under the transcriptional control of the 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 the 5' or 3' of a target mRNA, or in the target mRNA, or the first complementary region is 5' and its complementary sequence is 3' of the target mRNA.

[0055] 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.

[0056] The term "recombinant" refers to the artificial combination of two originally separate sequence segments, for example, by chemical synthesis or by the manipulation of separate nucleic acid segments through genetic engineering techniques.

[0057] 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.

[0058] The terms "recombinant DNA molecule", "recombinant DNA construct", "expression construct", "construct", and "recombinant construct" are used interchangeably herein. A recombinant DNA construct comprises an artificial combination of nucleic acid sequences, such as regulatory sequences and coding sequences that are not all found together in nature. For example, a recombinant DNA construct may comprise 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 which occurs naturally. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method that will be used to introduce the vector into a host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. The skilled artisan is fully aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate the host cell. Those skilled in the art will also recognize that different independent transformation events may result in different expression levels and patterns (Jones et al., (1985) EMBO J [Journal of the European Molecular Biology Association] 4: 2411-2418: De Almeida et al., (1989) Mol Gen Genetics [Molecular and General Genetics] 218: 78-86), and therefore typically multiple events are screened to obtain lines that display the desired expression levels and patterns. Such screening can be done by standard molecular biology assays, biochemical assays, and other assays, including Southern blot analysis, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme assays or activity assays, and / or phenotypic analysis.

[0059] 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 Zea mays 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 sequence (e.g., a polynucleotide sequence is obtained from maize, isolated, modified, and reintroduced into a corn plant). As used herein, "heterologous" with respect to a sequence may refer to a sequence that is derived from a different species, variant, exotic species, or, if derived from the same species, a sequence that 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 a 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 regulatory regions and / or polynucleotides provided herein can be entirely synthetic.

[0060] As used herein, the term "expression" refers to the production of a functional end product (e.g., mRNA, guide RNA, or protein) in either precursor or mature form.

[0061] "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).

[0062] "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.

[0063] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats ( C lustered R egularly I Interspaced S hort P alindromic RCRISPR loci refer to certain genetic loci encoding components of DNA cleavage systems, 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). CRISPR loci can consist of a CRISPR array, comprising short direct repeat sequences (CRISPR repeats) separated by short variable DNA sequences (called 'spacers'), which can be flanked by different Cas (CRISPR associated) genes.

[0064] As used herein, an "effector" or "effector protein" is a protein having an activity that includes recognition, binding, and / or cleavage of a polynucleotide target or nicking of a polynucleotide target. An effector or effector protein may also be an endonuclease. 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.

[0065] The term "Cas protein" refers to a protein that is produced by Cas ( CThe polypeptide encoded by a gene (associated with RISPR). Cas proteins include, but are not limited to, Cas9 protein, Cpf1 (Cas12) protein, C2c1 protein, C2c2 protein, C2c3 protein, 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 to all or part of a specific polynucleotide target sequence, and optionally nick all or part of a specific polynucleotide target sequence or cut all or part of a specific polynucleotide target sequence. The Cas endonuclease described herein comprises one or more nuclease domains. The endonuclease disclosed herein may include an endonuclease having one or more RuvC nuclease domains. 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.

[0066] A "Cas endonuclease" may include a domain that enables it to act as a double-strand break inducing agent. A "Cas endonuclease" may also include one or more modifications or mutations that eliminate or reduce its ability to cut double-stranded polynucleotides (dCas). In some aspects, a Cas endonuclease molecule may retain the ability to nick a single-stranded polynucleotide (e.g., a D10A mutation in a Cas9 endonuclease molecule) (nCas9).

[0067] "Functional fragments", "functionally equivalent fragments" and "functionally equivalent fragments" of Cas endonucleases are used interchangeably herein and refer to a portion or subsequence of a Cas endonuclease of the present disclosure, wherein the ability to recognize, bind and optionally unwind, nick or cleave (introduce single-strand or double-strand breaks) a target site is retained. A portion or subsequence of a Cas endonuclease may comprise a complete peptide or a partial (functional) peptide of any one of its domains, such as, for example, but not limited to, a complete functional portion of a Cas3 HD domain, a complete functional portion of a Cas3 helicase domain, a complete functional portion of a Cascade protein (such as, but not limited to, Cas5, Cas5d, Cas7 and Cas8b1).

[0068] The terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" of a Cas endonuclease or Cas effector protein are used interchangeably herein and refer to variants of the Cas effector proteins disclosed herein, in which the ability to recognize, bind to and optionally unwind, nick or cleave all or part of a target sequence is retained.

[0069] Cas endonucleases can also include multifunctional Cas endonucleases. The terms "multifunctional Cas endonucleases" and "multifunctional Cas endonuclease polypeptides" are used interchangeably herein and include reference to a single polypeptide having a Cas endonuclease function (comprising at least one protein domain that can be used as a Cas endonuclease) and at least another function, such as but not limited to, a function of forming a cascade (including at least a second protein domain that can form a cascade with other proteins). In one aspect, the multifunctional Cas endonuclease comprises at least one additional protein domain relative to those typical domains of the Cas endonuclease (inside upstream (5') or downstream (3', or in the interior 5' and 3', or any combination thereof).

[0070] 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.

[0071] 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 to, and optionally unwinding, nicking or cleaving all or part of a target sequence.

[0072] 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 is generally performed 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.

[0073] The term "without a 5'-cap" and the like is used herein to refer to RNAs that have, for example, a 5'-hydroxyl group instead of a 5'-cap. For example, such RNAs may be referred to as "uncapped RNAs". Because 5'-capped RNAs tend to be exported from the nucleus, uncapped RNAs may accumulate better in the nucleus after transcription. One or more RNA components herein are uncapped.

[0074] 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).

[0075] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a guide RNA, crRNA, or tracrRNA are used interchangeably herein and refer to a portion or subsequence of a guide RNA, crRNA, or tracrRNA of the present disclosure, respectively, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0076] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of a guide RNA, crRNA, or tracrRNA (respectively) are used interchangeably herein and refer to variants of a guide RNA, crRNA, or tracrRNA of the present disclosure, respectively, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0077] The terms "single guide RNA" and "sgRNA" are used interchangeably herein and refer to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) containing a variable targeting domain (linked to a tracr pairing sequence that hybridizes to the tracrRNA) and a tracrRNA (transactivating RNA). CR ISPR RNA) fusion. The single guide RNA may comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of a type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease, 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 to, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0078] 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.

[0079] 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., US 20150059010 A1 published on February 26, 2015), or any combination thereof.

[0080] 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 complex herein may comprise one or more Cas proteins of any of the known CRISPR systems (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) and one or more suitable polynucleotide components.

[0081] 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 to, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0082] The terms "target site", "target sequence", "target site sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", "genomic target locus", "target polynucleotide" and "protospacer" are used interchangeably herein and refer to polynucleotide sequences, for example, but not limited to, nucleotide sequences on a chromosome, episome, locus or any other DNA molecule in the genome of a cell (including chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA) at which the guide polynucleotide / Cas endonuclease complex can recognize, bind and optionally nick or cut. The target site can be an endogenous site in the genome of the cell, or alternatively, the target site can be heterologous to the cell and thus not naturally present in the genome of the cell, or the target site can be found in a heterogeneous genomic location compared to the location where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of the cell and is located at the endogenous or native location of the target sequence in the genome of the cell. "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the cell, but located at a different position in the genome of the cell (i.e., a non-endogenous or non-native position).

[0083] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (pre-spacer sequence) recognized (targeted) 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 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.

[0084] "Altered target site", "altered target sequence", "modified target site", "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein, which comprises at least one change when compared to a non-altered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0085] "Modified nucleotides" or "edited nucleotides" refer to a nucleotide sequence of interest that comprises at least one change when compared to its non-modified nucleotide sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0086] Methods for "modifying a target site" and "altering a target site" are used interchangeably herein and refer to methods for generating an altered target site.

[0087] As used herein, "donor DNA" is a DNA construct that includes a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.

[0088] 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 can be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template may further comprise homologous nucleotide sequences flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0089] The term "plant-optimized Cas endonuclease" herein refers to a Cas protein encoded by a nucleotide sequence that has been optimized for expression in a plant cell or plant, including a multifunctional Cas protein.

[0090] "Plant optimized nucleotide sequence encoding a Cas endonuclease," "plant optimized construct encoding a Cas endonuclease," and "plant optimized polynucleotide encoding a Cas endonuclease" are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, which has been optimized for expression in a plant cell or plant. Plants comprising a plant optimized Cas endonuclease include: plants comprising a nucleotide sequence encoding a Cas sequence, and / or plants comprising a Cas endonuclease protein. In one aspect, the plant optimized Cas endonuclease nucleotide sequence is a corn optimized, rice optimized, wheat optimized, soybean optimized, cotton optimized, or canola optimized Cas endonuclease.

[0091] The term "plant" generally includes whole plant, plant organ, plant tissue, seed, plant cell, seed and plant offspring. Plant cell includes but is not limited to cells derived from the following materials: seed, suspension culture, embryo, meristematic region, callus, leaf, root, bud, gametophyte, sporophyte, pollen and microspore. "Plant element" is intended to refer to whole plant or plant component, and can include differentiated and / or undifferentiated tissue, such as but not limited to plant tissue, part and cell type. In one embodiment, plant element is one of the following: whole plant, seedling, meristematic tissue, basic tissue, vascular tissue, epithelial tissue, seed, leaf, root, bud, stem, flower, fruit, runner, bulb, tuber, corm, asexual terminal branch, bud, young shoot, tumor tissue, and various forms of cell and culture (e.g., unicellular, protoplast, embryo, callus). The term "plant organ" refers to a group of tissues that form and function different parts of plant tissue or constitute 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 that can create other plants through the sexual or asexual reproduction 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.

[0092] "Progeny" includes any subsequent progeny of a plant.

[0093] 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, ears, cobs, shells, stems, roots, root tips, anthers, etc.), together with these parts themselves. Cereals mean mature seeds produced by commercial growers for purposes other than cultivation or propagation of species. The offspring, variants, and mutants of these regenerated plants are also included within the scope of the present invention, provided that these parts contain the introduced polynucleotides.

[0094] 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.

[0095] 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.

[0096] As used herein, a "male sterile plant" is a plant that does not produce viable or otherwise fertilized male gametes. As used herein, a "female sterile plant" is a plant that does not produce viable or otherwise fertilized female gametes. It should be appreciated that male sterile plants and female sterile plants can be female fertile and male fertile, respectively. It should be further appreciated that male fertile (but female sterile) plants can produce viable offspring when crossed with female fertile plants, and female fertile (but male sterile) plants can produce viable offspring when crossed with male fertile plants.

[0097] As used herein, the term "non-conventional yeast" refers to any yeast that is not a species of yeast of the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or the genus Schizosaccharomyces. (See "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols," K. Wolf, KD Breunig, G. Barth, eds., Springer-Verlag, Berlin, Germany, 2003).

[0098] In the context of the present disclosure, the term "hybridized" or "cross" refers to the fusion of gametes via pollination to produce offspring (i.e., cells, seeds, or plants). The term encompasses sexual crossing (one plant is 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).

[0099] The term "infiltration" refers to the phenomenon that the desired allele of the genetic locus is transferred from a genetic background to another genetic background. For example, the infiltration of the desired allele at the designated locus can be passed to at least one offspring plant via the sexual hybridization between two parental plants, wherein at least one parental plant has the desired allele in its genome. Alternatively, for example, allelic transmission can occur by the reorganization between two donor genomes, for example in fusion protoplasts, wherein at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, for example, the allele of the selection of the natural allele (mutated or edited) or marker or QTL of transgenic, modified.

[0100] The term "isoline" is a comparative term referring to organisms that are genetically identical but have been treated differently. In one example, two genetically identical corn plant embryos can be divided into two different groups, one group receiving a treatment (such as the introduction of a CRISPR-Cas effector endonuclease) and one group not receiving such a treatment as a control. Therefore, any phenotypic differences between the two groups may be attributed solely to the treatment, rather than to any inherent properties of the endogenous genetic composition of the plant.

[0101] "Introduced" 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 into the cell itself.

[0102] "Purpose polynucleotide" includes any nucleotide sequence encoding a protein or polypeptide that improves the desirability of a crop (i.e., a trait of agronomic interest). Purpose polynucleotide: includes, but is not limited to, polynucleotides encoding a trait important 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 agronomically or commercially important trait. 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.

[0103] "Complex trait loci" include genomic loci with multiple transgenes that are genetically linked to each other.

[0104] 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 use efficiency, 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 that does not contain the modifications derived from the methods and compositions herein.

[0105] "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.

[0106] As used herein, the terms "reduce," "less," "slower," and "increase," "faster," "enhanced," "greater" refer to a decrease or increase in a characteristic of a modified plant element or resulting plant 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 below an untreated control. More than, 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%, 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 over an untreated control.

[0107] As used herein, the term "preceded by" when referring to sequence position means that one sequence occurs upstream or 5' to another sequence.

[0108] The meanings of the abbreviations are as follows: “sec” means second, “min” means minute, “h” means hour, “d” means day, “uL” means microliter, “mL” means milliliter, “L” means liter, “uM” means micromole, “mM” means millimole, “M” means mole, “mmol” means millimole, “umole” or “umole” means micromole, “g” means gram, “ug” or “ug” means microgram, “ng” means nanogram, “U” means unit, “bp” means base pair, and “kb” means kilobase.

[0109] Double-strand break (DSB) inducing agents (DSB agents)

[0110] Double-strand breaks induced by double-strand break-inducing agents, such as endonucleases that cleave phosphodiester bonds in polynucleotide chains, can lead to the induction of DNA repair mechanisms, including the non-homologous end joining pathway as well as homologous recombination. Endonucleases include a range of different enzymes, including restriction endonucleases (see, e.g., Roberts et al., (2003) Nucleic Acids Res 1:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, ed. Craigie et al., (ASM Press, Washington, DC)), meganucleases (see, e.g., WO 2009 / 114321; Gao et al. (2010) Plant Journal 1:176-187), TAL effector nucleases or TALENs (see, e.g., US20110145940, Christian, M., T. Cermak, et al. 2010. Targeting DNA double-strand breaks with TAL effector nucleases. [Targeting DNA double-strand breaks with TAL effector nucleases] Genetics 186(2):757-61 and Boch et al., (2009), Science 326(5959):1509-12), zinc finger nucleases (see, e.g., Kim, YG, J. Cha et al. (1996). "Hybrid restriction enzymes: zinc finger fusions to FokI cleavage"), and CRISPR-Cas endonucleases (see, e.g., WO 2007 / 025097 application published on March 1, 2007).

[0111] In addition to double-strand break inducing agents, site-specific base conversions can also be achieved to engineer one or more nucleotide changes to create one or more EMEs described herein in a genome. These include, for example, site-specific base editing mediated by C·G to T·A or A·T to G·C base editing deaminases (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 cleavage. " Nature 533(7603) (2016): 420-4).

[0112] Any double-strand break or -nick or -modification inducing agent can be used in the methods described herein, including, for example but not limited to: Cas endonucleases, recombinases, TALENs, zinc finger nucleases, restriction endonucleases, meganucleases, and deaminases.

[0113] CRISPR system and Cas endonucleases

[0114] Methods and compositions for polynucleotide modification using CRISPR-associated (Cas) endonucleases are provided. Class I Cas endonucleases contain multi-subunit effector complexes (types I, III, and IV), while class 2 systems contain single-protein effectors (types II, V, and VI) (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; Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6): e60; and Koonin et al., 2017, Curr Opinion Microbiology 37: 67-78). In 2 types of type II systems, the Cas endonuclease and guide RNA (gRNA) compound work, the guide RNA guides the Cas endonuclease to cut the DNA target, so that the target can be recognized, combined and cut by the Cas endonuclease. The gRNA includes a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a variable targeting (VT) domain that hybridizes with the nucleotide sequence in the target DNA. In some aspects, the gRNA includes CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide the Cas endonuclease to its DNA target. The crRNA includes a spacer complementary to one strand of the double-stranded DNA target and a region that forms an RNA duplex with tracrRNA base pairing. In some aspects, the gRNA is a "single guide RNA" (sgRNA) comprising a synthetic fusion of crRNA and tracrRNA. In many systems, the polynucleotide complex guided by the Cas endonuclease recognizes a short nucleotide sequence adjacent to the target sequence (protospacer sequence), referred to as a "protospacer sequence adjacent motif" (PAM).

[0115] Examples of Cas endonucleases include, but are not limited to, Cas9 and Cpf1. Cas9 (formerly known as Cas5, Csn1, or Csx12) is a Class 2, Type II Cas endonuclease (Makarova et al., 2015, Nature Reviews Microbiology [Natural Microbiology Review] Volume 13: 1-15). The Cas9-gRNA complex can recognize the 3′ PAM sequence of the target site (NGG for Streptococcus pyogenes Cas9), thereby enabling the spacer of the guide RNA to invade the double-stranded DNA target, and if there is sufficient homology between the spacer and the protospacer sequence, a double-strand break is generated. The Cas9 endonuclease comprises a RuvC domain and an HNH domain that together produce a double-strand break, and the two can each produce a single-strand break. For the Streptococcus pyogenes Cas9 endonuclease, the double-strand break leaves a blunt end. Cpf1 is a class 2 type V Cas endonuclease and contains a nuclease RuvC domain but lacks a HNH domain (Yamane et al., 2016, Cell 165:949-962). Cpf1 endonuclease produces "sticky" overhangs.

[0116] Some uses of the Cas9-gRNA system at a genomic target site include, but are not limited to, insertion, deletion, substitution or modification of one or more nucleotides at a target site; modification or replacement of a target nucleotide sequence (such as a regulatory element); insertion of a target polynucleotide; gene knockout; gene knock-in; 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 by expressing an inverted repeat sequence as a target gene.

[0117] In some aspects, a "polynucleotide modification template" is provided, which comprises at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be at least one nucleotide substitution, addition, deletion or chemical modification. Optionally, the polynucleotide modification template can further comprise a homologous nucleotide sequence flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to the desired nucleotide sequence to be edited.

[0118] In some aspects, the target polynucleotide is inserted into the target site and provided as part of a "donor DNA" molecule. As used herein, "donor DNA" is a DNA construct including a target polynucleotide to be inserted into the target site of the Cas endonuclease. The donor DNA construct further comprises a first region and a second region of homology flanking the target polynucleotide. The first region and the second region of homology of the donor DNA share homology with the first and second genomic regions present in the target site of the cell or organism genome or flanking the target site, respectively. The donor DNA can be tethered with the guidance polynucleotide. The tethered donor DNA can allow co-localization of the target and the donor DNA, which can be used for genome editing, gene insertion and targeted genome regulation, and can also be used for targeting late mitotic cells, in which the function of the endogenous HR mechanism is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963). The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or region.

[0119] The process of editing the genomic sequence of the Cas9-gRNA double-strand break site using a modified template generally includes: providing a Cas9-gRNA complex to a host cell, the complex recognizes a target sequence in the host cell genome and is capable of inducing a single-strand or double-strand break in the genomic sequence, and optionally providing at least one polynucleotide modification template comprising at least one nucleotide change compared to the nucleotide sequence to be edited. The polynucleotide modification template may also include a nucleotide sequence flanking the at least one nucleotide change, wherein the flanking sequence is substantially homologous to the chromosomal region flanking the double-strand break. Genome editing using a double-strand break inducing agent (such as a Cas9-gRNA complex) has been described, for example, in the following: US 20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and WO 2016025131 published on February 18, 2016.

[0120] In order to promote the optimal expression and nuclear localization of eukaryotic cells, the gene comprising the Cas endonuclease can be optimized as described in WO2016186953 published on November 24, 2016, and then delivered to the cell as a DNA expression cassette by methods known in the art. In some aspects, the Cas endonuclease is provided as a polypeptide. In some aspects, the Cas endonuclease is provided as a polynucleotide encoding a polypeptide. In some aspects, the guide RNA is provided as a DNA molecule encoding one or more RNA molecules. In some aspects, the guide RNA is provided as RNA or chemically modified RNA. In some aspects, the Cas endonuclease protein and guide RNA are provided as ribonucleoprotein complexes (RNP).

[0121] Once double-strand breaks are induced in the genome, the cellular DNA repair machinery is activated to repair the breaks.

[0122] Double-strand break repair and polynucleotide modification

[0123] Double-strand break inducers, such as guide Cas endonucleases, can recognize, bind to DNA target sequences, and introduce single-strand (nick) or double-strand breaks. Once single-strand breaks or double-strand breaks are induced in DNA, the cell's DNA repair mechanism is activated to repair the break, for example, via non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes that lead to modifications at the target site. The most common repair mechanism used to join the ends of the breaks together 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 preserved by repair, but deletions, insertions, or other rearrangements (such as chromosome translocations) are possible (Siebert and Puchta, 2002 Plant Cell [Plant Cell] 14: 1121-31; Pacher et al., 2007 Genetics [Genetics] 175: 21-9). NHEJ is often error-prone and can introduce small mutations at the target site. In plants, NHEJ is often the major pathway for repairing DSBs; therefore, methods and compositions that improve the probability of HDR or HR in plants are needed.

[0124] As described by Podevin (Podevin, N., Davies, H.V., Hartung, F., Nogue, F. and Casacuberta, J.M. (2013) Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol. 31(6), 375-383), Hilscher (Hilscher, J., Burstmayr, H. and Stoger, E. (2016) Targeted modification of plant genomes for precision crop breeding. Biotechnol. J. 11, 1-14), and Pacher (Pacher and Puchta (2016), From classical mutagenesis to nuclease-based breeding-directing natural DNA repair for a natural end-product. The Plant Journal 90(4):819-833), three classes of site-directed nuclease-mediated genome modifications have been defined according to the categorization of ZFN activities and regulatory purposes by the New Technologies Working Group (NTWG; European Commission etc.) of the European Union (EU):

[0125] SDN1 encompasses the application of SDN without additional donor DNA or repair template. Thus, the outcome of the reaction clearly depends on the DSB repair pathway of the plant genome. Since the major DSB repair pathway is NHEJ, small insertions or deletions (SDN1a) may occur. In the case of tandem arrangement of SDNs, larger deletions (SDN1b) can be obtained. In addition, inversions (SDN1c) or translocations (SDN1d) can be generated by multiplexing the SDN1 approach (Hilscher et al., 2016).

[0126] SDN2 describes the use of SDN together with an additional DNA “polynucleotide modification template” to introduce small mutations in a controlled manner. Here, a template that is primarily homologous to the target sequence is provided as a substrate for HR-mediated DSB repair after the induction of one or two adjacent DSBs. This approach allows the introduction of small mutations that themselves could also occur naturally. Considering the size of plant genomes, small modifications of up to 20 nucleotides can be statistically considered GEs similar to naturally occurring genomic changes. Therefore, targeted genome modifications using ODM are also considered comparable to SDN2.

[0127] SDN3 describes the use of SDN together with additional "donor polynucleotides" or "donor DNA" to introduce large segments of foreign DNA at a predetermined locus, thereby adding or replacing genetic information. Mechanistically, this process relies on HR-mediated DSB repair (such as SDN2), and the distinction is arbitrary because the size of the inserted sequence can vary significantly.

[0128] Both SDN2 and SDN3 are types of homology-directed repair (HDR) of double-strand breaks in polynucleotides and involve the introduction of a heterologous polynucleotide as a template for repairing double-strand breaks (SDN2) or as the insertion of a new double-stranded polynucleotide at the site of the double-strand break (SDN3). SDN2 repair can be detected by the presence of one or a few nucleotide changes (mutations). SDN3 repair can be detected by the presence of new continuous heterologous polynucleotides.

[0129] The modification of target polynucleotide comprises following any one or more: the insertion of at least one nucleotide, the deletion of at least one nucleotide, the chemical change of at least one nucleotide, the replacement of at least one nucleotide or the sudden change of at least one nucleotide.In some respects, DNA repair mechanism causes the incomplete repair of double-strand break, causes the nucleotide at break site to change.In some respects, polynucleotide template can be provided to break site, wherein repair causes the template directed repair of break.In some respects, donor polynucleotide can be provided to break site, wherein repair causes the donor polynucleotide to be incorporated into break site.

[0130] In some aspects, the methods and compositions described herein improve the probability of non-NHEJ repair mechanism outcomes at DSBs.In one aspect, an increase in the HDR to NHEJ repair ratio is achieved.

[0131] Homologous directed repair and homologous recombination

[0132] Homologous directed repair (HDR) is a mechanism used in cells to repair double-stranded DNA and single-stranded DNA breaks. Homologous directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. [Annual Review of Biochemistry] 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 gaps (single-strand breaks) can occur via a different mechanism from HDR at double-strand breaks (Davis and Maizels. PNAS [Proceedings of the National Academy of Sciences of the United States] (0027-8424), 111 (10), pages E924-E932).

[0133] "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. The homologous region can have any length sufficient to promote homologous recombination at the target site of cleavage. For example, the length of the 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, 5-1500, 5-1600, 5-1700, 5-1800, 5-2000 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases so that the homologous region has enough homology to carry out homologous recombination with the corresponding genomic region. "Sufficient homology" means that two polynucleotide sequences have enough structural similarity 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.

[0134] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450 In some embodiments, the target site may be a target site having a length of 1-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or a region having a unit integer value in a range up to and including the total length of the target site. These ranges include each integer in the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by the percentage sequence identity on the complete comparison length 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 polynucleotide length, overall percentage sequence identity, and optionally any combination of conserved regions or local percentage sequence identity of continuous nucleotides, for example, enough homology can be described as a region of 75-150bp with at least 80% sequence identity with 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, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., eds. (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0135] DNA double-strand breaks can 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 repeat sequences 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).

[0136] The alteration of the genome of prokaryotic and eukaryotic cells or biological 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 150-200 bp 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.).

[0137] Improving the probability of HDR in DSB repair

[0138] Several approaches to promote repair of double-strand breaks via HDR are contemplated based on the following facts: (1) Cas9 has a high affinity for the substrates it cleaves and is slow to release (Richardson, C. et al. (2016) Nat. Biotechnol. [Nature Biotechnology] 34: 339-344); and (2) the inventors have observed that the mutational outcomes of polynucleotide cleavage are often non-random and reproducible (unpublished). The inventors envision retargeting polynucleotide double-strand break sites to provide multiple opportunities for DSB repair, promoting the occurrence of HDR (e.g., HR) relative to NHEJ. The inventors also envision that because recombinant gene intermediates involve 3' overhangs, additional single-strand breaks flanking the double-strand break site will produce unstable duplexes, thereby producing recombinant gene intermediates. In some cases, different endonucleases are used (e.g., from organisms or CRISPR loci from different sources, or engineered enzymes, or nickases).

[0139] In some aspects, the score or percentage of HR reads is greater than a comparison object, such as a control sample, a sample with NHEJ repair, or compared to the total mutation reads. In some aspects, the score or percentage of HR reads is greater than a control sample (without DSB agent). In some aspects, the score or percentage of HR reads is greater than the score or percentage of NHEJ reads. In some aspects, the score or percentage of HR reads is greater than the score or percentage of total mutation reads (NHEJ+HR).

[0140] In some aspects, the fraction of HR reads relative to a comparator is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, between 10 and 15, 15, between 15 and 20, 20, between 20 and 25, 25, between 25 and 30, 30, between 30 and 40, 40, between 40 and 50, 50, between 50 and 60, 60, between 60 and 70, 70, between 70 and 80, 80, between 80 and 90, 90, between 90 and 100, 100, between 100 and 125, 125, between 125 and 150, greater than 150, or infinity.

[0141] In some aspects, the percentage of HR reads relative to a comparator is at least 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%, 28%, 29%, 20%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% greater.

[0142] In some aspects, the percentage of HR reads is greater than zero.

[0143] In one aspect of the method, by any method or composition, such as but not limited to Cas endonuclease and guide RNA, create, repair and repeatedly cut double-strand breaks. In short, DSB inducing agent (for example, Cas endonuclease and the first guide RNA) identifies, binds and cuts the target polynucleotide. Create and repair the first double-strand break. In some aspects, the repair causes a change in the target site polynucleotide sequence (such as but not limited to the insertion of nucleotides, the deletion of nucleotides or the replacement of nucleotides). In some aspects, a repair template is provided for a specific target polynucleotide repair composition result. In this case, the flank of the repair template is a reverse target site (PAM inside). Introduce the second guide RNA, which is complementary to the mutation created by the first double-strand break repair. In some aspects, the DSB repair composition result is determined by introducing a donor polynucleotide template or inserting, and the second guide RNA is designed to complement the target sequence result determined therefrom. In some aspects, the second guide RNA is designed to complement the repair mutation most often created. In some aspects, the second guide RNA is designed to complement the required DNA repair result. In some aspects, a library of a second guide RNA complementary to all possible mutations of the target site is designed. One or more mutations created by the first double-strand break repair can be known or predicted in a bioinformatics manner. The second guide RNA and Cas endonuclease (re-provided or with the same Cas endonuclease present for the first DSB) synergize to create a second double-strand break at the same site (within the target recognition sequence of the Cas endonuclease / first guide RNA complex). In some aspects, instead of creating the second guide RNA and Cas endonuclease of the second DSB, another DSB inducing agent can be introduced. Compared with the repair of the first DSB, the HDR repair probability of the second DSB is higher than the NHEJ repair probability (i.e., the probability of HDR increases, or the frequency of HDR increases, or the ratio of HDR to NHEJ increases). Generally, there is a subsequent cutting at the previous cleavage site, and in some aspects, the subsequent cutting can be achieved by introducing another Cas endonuclease / gRNA complex. Continuing to cut in a sequential manner increases the frequency of HDR as a DSB repair mechanism.

[0144] In one aspect of the method, by any method or composition, such as but not limited to Cas endonuclease and guide RNA, create, repair and recursively cut double-strand breaks.In short, DSB inducer (e.g., Cas endonuclease and first guide RNA) recognizes, binds and cuts target polynucleotide.The first guide RNA is provided as a DNA sequence on a plasmid, and the plasmid further comprises a spacer sequence.In some aspects, the DNA encoding gRNA is operably connected to a regulatory expression element.Create and repair the first double-strand break.The composition of the repaired target polynucleotide is used as the basis for the mutation generated by Cas editing the spacer on the plasmid comprising gRNA DNA and spacer.The spacer composition of the mutation guides the generation of the second gRNA, which is complementary to the sequence of the targeted polynucleotide repaired by the first DSB, and induces the second double-strand break at the target site by Cas endonuclease and the second gRNA.The cycle can then be repeated, and the sequence of the newly repaired second DSB is then used as a template for the composition of the third gRNA complementary to the sequence of the second DSB polynucleotide repaired, and so on. In this manner, a cycle of DSB generation and repair occurs, with each subsequent repair after the first having a higher probability of being repaired via HDR than via NHEJ. The process can be stopped by any of a variety of methods, including, but not limited to: titration of reagent availability, inducing a mutation in the region of the gRNA DNA expression construct that renders the expression cassette or transcribed gRNA nonfunctional, which can optionally be an inducible or repressible external factor, or via the introduction of another molecule.

[0145] In one aspect of the method, a nick adjacent to a double-strand break on a target polynucleotide is created (cutting double-stranded DNA only on one of two phosphate backbones). In a variation in this regard, a single nick is created. In a variation in this regard, two nicks are created. In a variation in this regard, two nicks are created, each flanking the two sides of the DSB. In one embodiment, a double-strand break is created by a Cas endonuclease, and one or more nicks are created by different molecules (e.g., molecules derived from different organisms, or lacking double-strand break creation functions but having Cas endonucleases (e.g., nCas9) with nickase activity). Due to the presence of one or more adjacent nicks, the probability of the double-strand break repair of DSB at the target site being repaired by HDR is higher than the probability of being repaired by NHEJ, or compared with the DSB without one or more nicks adjacent to the DSB at the same locus, the frequency of HDR is higher. In some aspects, the length of the distance between the nick and the DSB site is 10 base pairs, between 10 and 20 base pairs, 20 base pairs, between 20 and 30 base pairs, 30 base pairs, between 30 and 40 base pairs, 40 base pairs, between 40 and 50 base pairs, 50 base pairs, between 50 and 60 base pairs, 60 base pairs, between 60 and 70 base pairs, 70 base pairs, between 70 and 80 base pairs, 80 base pairs, between 80 and 90 base pairs, 90 base pairs, between 90 and 100 base pairs, 100 base pairs, between 100 and 110 base pairs, 110 base pairs, between 110 and 120 base pairs, or greater than 120 base pairs.

[0146] In addition to increasing the probability of an HDR repair mechanism outcome, other DNA repair outcomes contemplated for improvement using the methods described herein include gene targeting, gene editing, gene dropout, gene exchange (deletion plus insertion), and promoter exchange (deletion plus insertion).

[0147] Gene targeting

[0148] The compositions and methods described herein can be used for gene targeting.

[0149] Typically, DNA targeting can be performed by cutting one or both strands at a specific polynucleotide sequence in a cell with a Cas endonuclease associated with a suitable guide polynucleotide component. Once single-strand breaks or double-strand breaks are induced in the DNA, the cell's DNA repair machinery is activated to repair the breaks via non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes that result in modifications at the target site.

[0150] The length of the DNA sequence at the target site can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 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 as 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 each other to produce a blunt-end cleavage, or in other cases, the nicks can be staggered to produce single-stranded overhangs, also known as "sticky ends", which can be either 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 the active variant retains biological activity and is therefore capable of being recognized and cleaved by a Cas endonuclease.

[0151] Assays that measure single-strand 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.

[0152] 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. This method can optionally be characterized as a multiplex method. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten or more target sites can be targeted simultaneously. Multiplexing methods are 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.

[0153] Gene Editing

[0154] The process of editing a genomic sequence by combining DSB and a modification template generally includes: introducing a DSB inducer or a nucleic acid encoding a DSB inducer (recognizing a target sequence in a chromosomal sequence and being able to induce 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 also include 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 a DSB inducer (such as a Cas-gRNA complex) has been described, for example, in US 20150082478 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.

[0155] Some uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US 20150082478 A1 published on March 19, 2015, WO 2015026886 published on February 26, 2015, and US 20150059010 published on February 26, 2015) 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 withdrawal, 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.

[0156] 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 the 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, are preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in protein characteristics, 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 containing a target site.

[0157] This article describes a method for using the cut-ready Cascade ( C leavage R easy CMethods for genome editing using a cleavage-ready Cascade (crCascade) complex. After characterizing the guide RNA and PAM sequences, the components of the cleavage-ready Cascade (crCascade) complex and the associated CRISPR RNA (crRNA) can be used to modify chromosomal DNA in other organisms, including plants. To promote optimal expression and nuclear localization (for eukaryotic cells), the gene containing crCascade 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 crCascade complex can also be delivered as RNA (with or without modifications that protect the RNA from degradation) or as capped or uncapped mRNA (Zhang, Y. et al., 2016, Nat. Commun. [Natural Communications] 7: 12617) or Cas protein-guided polynucleotide complex (published in WO 2017070032 on April 27, 2017), or any combination thereof. In addition, one or more portions of the crCascade complex and crRNA can be expressed from a DNA construct, while the other components are delivered as RNA (with or without modifications that protect the RNA from degradation) or as capped or uncapped mRNA (Zhang et al. 2016 Nat. Commun. [Nature Communications] 7: 12617) or Cas protein-guided polynucleotide complexes (published in WO 2017070032 on April 27, 2017), or any combination thereof. For in vivo production of crRNA, tRNA-derived elements can also be used to recruit endogenous RNases to cleave crRNA transcripts into mature forms capable of directing the crCascade complex to its DNA target site, for example, as described in WO2017105991 published on June 22, 2017. The crCascade nickase complex can be used alone or in concert to produce single or multiple DNA nicks on one or both DNA strands. In addition, the cleavage activity of Cas endonucleases can be inactivated by changing 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 RNA-guided helicases that can be used to enhance homology-directed repair, induce transcriptional activation, or remodel local DNA structures. Moreover, the activities of both the 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.

[0158] The transcription direction of tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (such as 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.

[0159] 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-ready Cascade (crCascade) complex results in degradation of the random PAM library, the crCascade complex can be converted into a nickase by mutagenesis of key residues or by inactivating the ATPase-dependent helicase activity by assembling the reaction in the absence of ATP, 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 pro-spacer targets, which can be captured and sequenced to check for PAM sequences that support cleavage by the respective crCascade complexes.

[0160] In one embodiment, the invention features a method for modifying a target site in the genome of a cell, the method comprising introducing at least one Cas endonuclease and a guide RNA into the cell, and identifying at least one cell having a modification at the target site.

[0161] 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 the at least one nucleotide to be edited and the genomic target site.

[0162] 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.

[0163] 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.

[0164] The method for editing a nucleotide sequence in the genome of a cell may be a method by which the function of a non-functional gene product is restored without using an exogenous selective marker.

[0165] 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.

[0166] 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.

[0167] A variety of methods and compositions can be used to produce cells or organisms with a target polynucleotide of interest inserted into a target site via the activity of a CRISPR-Cas system component as described herein. In one method described herein, a target polynucleotide is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" is a DNA construct comprising a target polynucleotide to be inserted into a target site of a Cas endonuclease. The donor DNA construct further comprises a first region and a second region of homology flanking the target polynucleotide. The first and second regions of the homology of the donor DNA share homology with the first and second genomic regions present in the target site of a cell or organism genome or flanking the target site, respectively.

[0168] The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, which 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 the endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Nature Methods] Vol. 10: 957-963).

[0169] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450 In some embodiments, the target site may be a target site having a length of 1-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or a region having a unit integer value in a range up to and including the total length of the target site. These ranges include each integer in the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by the percentage sequence identity on the complete comparison length 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 polynucleotide length, overall percentage sequence identity, and optionally any combination of conserved regions or local percentage sequence identity of continuous nucleotides, for example, enough homology can be described as a region of 75-150bp with at least 80% sequence identity with 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, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., eds. (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0170] 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 homologous sequences are available, 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).

[0171] 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.

[0172] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of a target genomic nucleotide sequence. (See also US 20150082478 published on March 19, 2015 and WO 2015026886 published on February 26, 2015).

[0173] 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 01, 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.

[0174] 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 use of double-strand break inducing agents to introduce the target gene. 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 US 20130263324 published on October 03, 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 all two transgenes. In the offspring from these F1 (F2 or BC1), 1 / 500 of the offspring will have two different transgenes recombined on the same chromosome. The composite locus can then be bred to a single genetic locus with all two transgenic traits. This process can be repeated to stack as many traits as possible.

[0175] Further uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US 20150082478 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.

[0176] 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 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 a QTL encompasses or is closely linked to one or more genes that affect the trait under consideration. "Alleles of a QTL" may include multiple genes or other genetic factors, such as haplotypes, in a continuous genomic region or linkage group. The alleles of a QTL may represent haplotypes within a specified window, wherein the window is a continuous genomic region that can be defined and tracked with a set of one or more polymorphic markers. A haplotype may be specified as being defined by a unique fingerprint of the alleles of each marker within the window.

[0177] Recombinant constructs and transformation of cells

[0178] The guiding polynucleotides, Cas endonucleases, polynucleotide modification templates, donor DNA, guiding polynucleotide / Cas endonuclease systems, and any combination thereof (optionally further comprising one or more polynucleotides of interest) disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein.

[0179] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are more fully described in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described below.

[0180] Vectors and constructs include circular plasmids and linear polynucleotides comprising a polynucleotide of interest, and optionally include linkers, adapters, other components for regulation or analysis. In some examples, the recognition site and / or target site may be contained within an intron, coding sequence, 5'UTR, 3'UTR, and / or regulatory region.

[0181] Components for expressing and utilizing CRISPR-Cas systems in prokaryotic and eukaryotic cells

[0182] The present invention also provides expression constructs for expressing a guide RNA / Cas system in a prokaryotic or eukaryotic cell / organism that is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or part of a target sequence.

[0183] In one embodiment, the expression construct of the present invention comprises a promoter operably linked to a nucleotide sequence encoding a Cas gene (or plant-optimized, including a Cas endonuclease gene as described herein) and a promoter operably linked to a guide RNA of the present disclosure. The promoter is capable of driving the expression of an operably linked nucleotide sequence in a prokaryotic or eukaryotic cell / organism.

[0184] The nucleotide sequence modifications of the guide polynucleotide, VT domain and / or CER domain can be selected from, but not limited to, the group consisting of: a 5' cap, a 3' poly(A) tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a protein binding site, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotides, 2'-fluoro U nucleotides; 2'-O-methyl RNA nucleotides, a phosphorothioate bond, a linkage to a cholesterol molecule, a linkage to a polyethylene glycol molecule, a linkage to a spacer 18 molecule, a 5' to 3' covalent linkage, or any combination thereof. The modifications can produce at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of: modified or regulated stability, subcellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity to complementary target sequences, modified resistance to cellular degradation, and increased cellular permeability.

[0185] Methods for expressing RNA components (such as gRNA) for Cas9-mediated DNA targeting in eukaryotic cells have used RNA polymerase III (Pol III) promoters, which allow RNA transcription with precisely defined unmodified 5'- and 3'-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3: e161). This strategy has been successfully applied in cells of several different species, including corn and soybean (US 20150082478 published on March 19, 2015). Methods for expressing RNA components that do not have a 5' cap have been described (WO 2016 / 025131 published on February 18, 2016).

[0186] Different methods and compositions can be used to obtain cells or organisms having a target polynucleotide inserted into a target site for a Cas endonuclease. Such methods can use homologous recombination (HR) to provide integration of the target polynucleotide at the target site. In a method described herein, the target polynucleotide is introduced into an organism cell via a donor DNA construct.

[0187] The donor DNA construct further comprises a first region and a second region of homology flanking the polynucleotide of interest. The first region and the second region of homology of the donor DNA share homology with the first and second genomic regions present in or flanking the target site of the genome of a cell or organism, respectively.

[0188] The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, which 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 the endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Nature Methods] Vol. 10: 957-963).

[0189] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450 In some embodiments, the target site may be a target site having a length of 1-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or a region having a unit integer value in a range up to and including the total length of the target site. These ranges include each integer in the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by the percent sequence identity over the entire compared length of the two polynucleotides, including at least about 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%, 98% to 99%, 99%, 99% to 100% or 100% percent sequence identity. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example, sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., eds. (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0190] The structural similarity between a given genomic region and a corresponding homologous region found on the donor DNA can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of homology or sequence identity shared by a "homologous region" of the donor DNA and a "genomic region" of the organism's genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination.

[0191] The regions of homology on the donor DNA may have homology to any sequence flanking the target site. Although in some cases, the regions of homology share significant sequence homology with genomic sequences immediately flanking the target site, it should be recognized that the regions of homology can be designed to have sufficient homology to regions that may be closer to the 5' or 3' of the target site. The regions of homology may also have homology to fragments of the target site as well as downstream genomic regions.

[0192] In one embodiment, the first region of homology further comprises a first segment in the target site, and the second region of homology comprises a second segment in the target site, wherein the first segment and the second segment are different.

[0193] Target polynucleotide

[0194] Target polynucleotides are further described herein, and include polynucleotides that reflect those commercial markets and benefits related to crop development. Target crops and markets change, and as developing countries open international markets, new crops and technologies will also appear. In addition, as our understanding of agronomic traits and characteristics (such as yield and heterosis increase) gradually deepens, the selection of genes for genetic engineering will change accordingly.

[0195] General categories of polynucleotides of interest include, for example, those genes of interest that are involved in information (such as zinc fingers), those genes that are involved in communication (such as kinases), and those genes that are involved in housekeeping (such as heat shock proteins). More specific polynucleotides of interest include, but are not limited to, genes that are involved in traits of agronomic importance, such as, but not limited to, crop yield, grain quality, crop nutritional content, starch and carbohydrate quality and quantity, as well as genes that affect grain size, sucrose loading, protein quality and quantity, nitrogen fixation and / or nitrogen utilization, fatty acid and oil composition, genes encoding proteins that confer resistance to abiotic stresses (e.g., drought, nitrogen, temperature, salinity, toxic metals, or trace elements), or those that confer resistance to toxins (e.g., pesticides and herbicides), genes encoding proteins that confer resistance to biotic stresses (e.g., fungal, viral, bacterial, insect and nematode attack and the development of diseases associated with these organisms).

[0196] In addition to using traditional breeding methods, agronomically important traits such as oil, starch, and protein content can also be altered genetically. Modifications include increasing the content of oleic acid, saturated and unsaturated oils, increasing the levels of lysine and sulfur, providing essential amino acids, and also modifications to starch. Protein modifications of hordothionin are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0197] The polynucleotide sequence of interest can encode proteins that are involved in providing disease or pest resistance. "Disease resistance" or "pest resistance" is intended to be a plant that avoids the occurrence of harmful symptoms that are a consequence of plant-pathogen interactions. Pest resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Disease resistance genes and insect resistance genes, such as lysozymes or cecropins for antibacterial protection, or proteins for antifungal protection, such as defensins, glucanases, or chitinases, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects, are examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as resistance to fumonisins (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); etc. Insect resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); etc.

[0198] "Herbicide resistance proteins" or proteins expressed by "herbicide resistance encoding nucleic acid molecules" include proteins that confer to cells the ability to tolerate higher concentrations of herbicides compared to cells that do not express the protein, or confer to cells the ability to tolerate a certain concentration of herbicides for a longer period of time compared to cells that do not express the protein. The herbicide resistance trait can be introduced into plants by genes encoding resistance to herbicides that inhibit acetolactate synthase (ALS, also known as acetohydroxyacid synthase, AHAS) (particularly sulfonylurea (UK: sulfonylurea (sulphonylurea)) herbicides), genes encoding resistance to herbicides that inhibit glutamine synthase (e.g., phosphinothricin or basta) (e.g., bar genes), genes encoding resistance to glyphosate (e.g., EPSP synthase genes and GAT genes), genes encoding resistance to HPPD inhibitors (e.g., HPPD genes), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutant encodes resistance to the herbicide chlorsulfuron.

[0199] In addition, it is recognized that the target polynucleotide can also include an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) of the gene sequence targeted for the purpose. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. The antisense sequence can be modified as long as the sequence hybridizes with the corresponding mRNA and interferes with the expression of the corresponding mRNA. In this way, an antisense construct with 70%, 80% or 85% sequence identity to the corresponding antisense sequence can be used. In addition, the part of the antisense nucleotide can be used to destroy the expression of the target gene. Usually, a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more nucleotides can be used.

[0200] In addition, the target polynucleotide can also be used in a sense orientation to suppress the expression of endogenous genes in plants. The method of using polynucleotides in a sense orientation to suppress gene expression in plants is known in the art. These methods generally relate to transformed plants with a DNA construct comprising a promoter, which is operably connected to at least a portion of the nucleotide sequence of the transcript corresponding to the endogenous gene, driving the expression in the plant. Generally, such nucleotide sequences have substantial sequence identity with the sequence of the transcript of the endogenous gene, generally greater than about 65% sequence identity, about 85% sequence identity or greater than about 95% sequence identity. Referring to U.S. Patent Nos. 5,283,184 and 5,034,323.

[0201] The target polynucleotide can also be a phenotypic marker. Phenotypic markers are screenable or selective markers, which include visual markers and selective markers, whether it is a positive or negative selective marker. Any phenotypic marker can be used. Specifically, selectable or screenable markers include DNA segments that allow people to identify or select molecules or cells that contain it or select it under specific conditions. These markers can encode activity, such as but not limited to the production of RNA, peptides or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc.

[0202] Examples of selectable markers include, but are not limited to, DNA segments comprising restriction endonuclease sites; DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT); DNA segments encoding products that are inherently deficient in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that are easily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); generating new primer sites for PCR (e.g., juxtaposition of two DNA sequences that were not previously juxtaposed), comprising DNA sequences that are inactivated or activated by restriction endonucleases or other DNA modifying enzymes, chemicals, etc.; and comprising DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0203] Additional selectable markers include genes that confer resistance to herbicidal compounds such as sulfonylureas, glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See, for example, acetolactate synthase (ALS) for resistance to sulfonylureas, imidazolinones, triazolopyrimidinesulfonamides, pyrimidine salicylates, and sulfonylaminocarbonyl-triazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110); glyphosate resistance 5-enolpyruvylshikimate-3-phosphate (EPSPS) (Saroha et al., 1998, J. Plant Biochemistry & Biotechnology 7: 65-72);

[0204] Target polynucleotides include genes that are stacked or used in combination with other traits (such as, but not limited to, herbicide resistance or any other trait described herein). Target polynucleotides and / or traits can be stacked together in a complex trait locus, as described in US 20130263324 published on October 3, 2013 and WO / 2013 / 112686 published on August 1, 2013.

[0205] The polypeptide of interest includes a protein or polypeptide encoded by a polynucleotide of interest described herein.

[0206] Further provided is a method for identifying at least one plant cell, which contains the target polynucleotide integrated at the target site in its genome. A variety of methods can be used to identify those plant cells inserted into the genome at or near the target site. Such methods can be considered to directly analyze the target sequence to detect any change in the target sequence, including but not limited to PCR method, sequencing method, nuclease digestion, Southern blotting, and any combination thereof. See, for example, US20090133152 announced on May 21, 2009. The method also includes reobtaining plants from plant cells containing the target polynucleotide integrated into its genome. The plant can be sterile or fertile. It should be appreciated that any target polynucleotide can be provided, which is integrated into the genome of the plant at the target site and expressed in the plant.

[0207] Optimization of sequences for expression in plants

[0208] Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498. Additional sequence modifications are known to enhance gene expression in plant hosts. For example, these sequence modifications include elimination of: one or more sequences encoding false polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. The GC content of the sequence can be adjusted to an average level for a given plant host calculated by reference to known genes expressed in host plant cells. When possible, the sequence is modified to avoid the presence of one or more predicted hairpin secondary mRNA structures. Therefore, the "plant-optimized nucleotide sequence" of the present disclosure includes one or more such sequence modifications.

[0209] Expression elements

[0210] Any polynucleotide encoding Cas proteins, other CRISPR system components, or other polynucleotides disclosed herein may be functionally linked to heterologous expression elements to promote transcription or regulation in host cells. Such expression elements include, but are not limited to, promoters, leaders, introns, and terminators. An expression element may be "minimal" - meaning a shorter sequence derived from a natural source that still acts as an expression regulator or modifier. Alternatively, an expression element may be "optimized" - meaning that its polynucleotide sequence has been changed from its native state so that it functions with more desirable characteristics in a specific host cell (for example, but not limited to, a bacterial promoter may be "corn optimized" to improve its expression in corn plants). Alternatively, an expression element may be "synthetic" - meaning that it is designed by computer and synthesized for use in a host cell. Synthetic expression elements may be fully synthetic or partially synthetic (fragments comprising naturally occurring polynucleotide sequences).

[0211] Certain promoters have been shown to be able to direct RNA synthesis at a higher rate than other promoters. These are referred to as "strong promoters". Certain other promoters have been shown to direct RNA synthesis only at higher levels in specific types of cells or tissues, and if the promoter preferentially directs RNA synthesis in certain tissues but also at reduced levels in other tissues, it is often referred to as a "tissue-specific promoter" or "tissue-preferential promoter".

[0212] Plant promoters include promoters that are capable of initiating transcription in plant cells. For a review of plant promoters, see Potenza et al., 2004 In vitro Cell Dev Biol 40: 1-22; Porto et al., 2014, Molecular Biotechnology (2014), 56(1), 38-49.

[0213] Constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; ALS promoter (U.S. Pat. No. 5,659,026), etc.

[0214] Tissue-preferred promoters can be used to target enhanced expression in specific plant tissues. Tissue-preferred promoters include, for example, WO 2013103367, published on July 11, 2013, Kawamata et al., (1997) Plant Cell Physiol [Plant Cell Physiology] 38: 792-803; Hansen et al., (1997) Mol Gen Genet [Molecular and General Genetics] 254: 337-43; Russell et al., (1997) Transgenic Res [Transgenic Research] 6: 157-68; Rinehart et al., (1996) Plant Physiol [Plant Physiology] 112: 1331-41; Van Camp et al., (1996) Plant Physiol. [Plant Physiology] 112: 525-35; Canevascini et al., (1996) Plant Physiol. [Plant Physiology] 112: 525-35; Physiol. [Plant Physiology] 112: 513-524; Lam, (1994) Results Probl Cell Differ [Results and Problems in Cell Differentiation] 20: 181-96; and Guevara-Garcia et al., (1993) Plant J. [Plant Journal] 4: 495-505. Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al., (1958) EMBO J [Journal of the European Molecular Biology Association] 4: 2723-9; Timko et al., (1988) Nature 318: 57-8.Root-preferred promoters include, for example, Hire et al., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); Bogusz et al., (1990) Plant Cell 3:11-23 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); Cell 2:633-41 (root-specific promoter isolated from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (rolC and rolD root-inducible genes of Agrobacterium rhizogenes); Teeri et al., (1989) EMBO J 8:343-50 (TR1′ and TR2′ genes induced by wounding of Agrobacterium); VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al., (1994) Plant Mol Biol 25:681-91); phaseolin gene (Murai et al., (1983) Science 23:476-82; Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

[0215] Seed-preferred promoters include both seed-specific promoters active during seed development and seed germination promoters active during seed germination. See Thompson et al., (1989) BioEssays [Biological Analysis] 10: 108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (inositol-1-phosphate synthase); and, for example, those disclosed in WO 2000011177 published on March 2, 2000 and U.S. Patent 6,225,529. For dicotyledons, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, wax, contractin 1, contractin 2, globulin 1, oleosin, and nucl. See also WO 2000012733, published March 9, 2000, which discloses seed-preferred promoters from END1 and END2 genes.

[0216] Chemical inducible (regulatable) promoters can be used to regulate gene expression in prokaryotic and eukaryotic cells or organisms by applying exogenous chemical regulators. The promoter can be a chemical inducible promoter in the case of application of a chemical to induce gene expression, or a chemical repressible promoter in the case of application of a chemical to repress gene expression. Chemical inducible promoters include, but are not limited to: the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol [Plant Cell Physiology] 38: 568-77), the maize GST promoter activated by hydrophobic electrophilic compounds used as pre-emergence herbicides (GST-II-27, WO 1993001294 published on January 21, 1993), and the tobacco PR-1a promoter activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem [Bioscience Biotechnology Biochemistry] 68: 803-7). Other chemically-regulated promoters include steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters (Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al., (1998) Plant J 14:247-257); tetracycline-inducible promoters and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156).

[0217] Pathogen-inducible promoters that are induced upon infection by pathogens include, but are not limited to, promoters that regulate the expression of PR proteins, SAR proteins, β-1,3-glucanase, chitinase, and the like.

[0218] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17: 287-91). Those skilled in the art are familiar with protocols for simulating stress conditions (such as drought, osmotic stress, salt stress, and temperature stress) and evaluating stress tolerance in plants that have been subjected to simulated or naturally occurring stress conditions.

[0219] Another example of an inducible promoter useful in plant cells is the ZmCAS1 promoter, described in US 20130312137 published on November 21, 2013.

[0220] New promoters of different types that are useful in plant cells are continually being discovered; many examples can be found in the compilation by Okamuro and Goldberg, (1989) The Biochemistry of Plants, Vol. 115, ed. Stumpf and Conn (New York, NY: Academic Press), pp. 1-82.

[0221] Introducing system components into cells

[0222] The methods described herein do not depend on a specific 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 cell or a 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) into the cell.

[0223] 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.

[0224] For example, a guide polynucleotide (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 indirectly introduced into cells by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding a guide RNA (or crRNA + tracrRNA), which is operably connected to a specific promoter capable of transcribing the guide RNA (or crRNA + tracrRNA) in the cell. The specific promoter can be, but is not limited to, an RNA polymerase III promoter, which allows 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 include heat shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the guide RNA.

[0225] Cas endonucleases herein, such as Cas endonucleases described herein, can be introduced into cells by directly introducing Cas polypeptides themselves (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 published 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 connected to nucleotide sequences encoding Cas endonucleases.

[0226] 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 (such as plant cells).

[0227] 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 a Cas endonuclease and a target site, the donor DNA is inserted into the genome of the transformed plant.

[0228] Direct delivery of any one of the guided Cas system components can be accompanied by direct delivery (co-delivery) of other mRNAs that can promote 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 restore the function of non-functional gene products without using exogenous selectable markers to achieve cell selection and enrichment, as described in WO2017070032 published on April 27, 2017.

[0229] Introducing the guide RNA / Cas endonuclease complex described herein (representing the cleavage-ready cascade described herein) into a cell includes introducing the components of the complex into the cell individually or in combination, and directly (delivered 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 (targeted for genome modification as described herein).

[0230] Plant cells differ from human and animal cells in that plant cells contain a plant cell wall, which may serve as a barrier to the direct delivery of ribonucleoproteins and / or the direct delivery of these components.

[0231] Direct delivery of ribonucleoproteins containing Cas endonuclease proteins and guide RNAs into plant cells can be achieved by particle-mediated delivery (particle bombardment). Based on the experiments described herein, technicians can now envision that any other direct delivery methods (such as but not limited to polyethylene glycol (PEG)-mediated transfection of protoplasts, electroporation, cell-penetrating peptides or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery) can be successfully used to deliver RGEN ribonucleoproteins into plant cells.

[0232] Direct delivery of ribonucleoprotein allows genome editing at the target site in the genome of the cell, after which the complex can be rapidly degraded, and only the complex is allowed to exist briefly in the cell. This short-lived presence of the complex may result in a reduction in off-target effects. In contrast, delivery of components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result in constant expression from these plasmids, which can promote off-target cutting in some cases (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).

[0233] Direct delivery can be achieved by combining any one component of the guide RNA / Cas endonuclease complex (representing the cleavage-ready cascade described herein), such as at least one guide RNA, at least one Cas protein, and optionally at least one additional protein, with a particle delivery matrix comprising microparticles, such as but not limited to gold particles, tungsten particles, and silicon carbide whisker particles (see also WO2017070032 published on April 27, 2017).

[0234] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

[0235] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein and at least one protein subunit of cascade forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

[0236] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and Cas endonuclease protein forming the guide RNA / Cas endonuclease complex (cleavage-ready cascade) and at least one protein subunit of the cascade are preassembled in vitro and introduced into the cell as a ribonucleotide-protein complex.

[0237] 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.Code: 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 [Proceedings of the National Academy of Sciences of the United States of America] 85:4305-9 (corn); Klein et al., (1988) Biotechnology [Biotechnology] 6:559-63 (corn); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol [Plant Physiol] 91:440-4 (corn); Fromm et al., (1990) Biotechnology [Biotechnology] 8:833-9 (corn); 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) in The Experimental Manipulation of Ovule Tissues, Chapman et al., eds. (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).

[0238] Alternatively, polynucleotides can be introduced into 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 synthesized polypeptide 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.

[0239] 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.

[0240] 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 (such as cell penetrating peptides and nanocarriers) of the guided Cas system (proteins and / or nucleic acids). See also US 20110035836 published on February 10, 2011 and EP 2821486 A1 published on January 7, 2015.

[0241] 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.

[0242] "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 offspring. "Transient transformation" is intended to mean that a polynucleotide is introduced into the organism and is not integrated into the genome of the organism, or that a polypeptide is introduced into the organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0243] 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.

[0244] Cells and Organisms

[0245] The polynucleotides and polypeptides disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, mammal, bacteria, protist, fungi, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. In some aspects, the cells of an organism are reproductive cells, somatic cells, meiotic cells, mitotic cells, stem cells, or pluripotent stem cells. Any cell from any organism can be used with the compositions and methods described herein, including monocots and dicots and plant elements.

[0246] Animal cells

[0247] The polynucleotides and polypeptides disclosed herein can be introduced into animal cells. Animal cells can include, but are not limited to, organisms of the following phyla, including Chordata, Arthropoda, Mollusca, Annelida, Coelenterate, or Echinodermata; organisms of the following classes, including mammals, insects, birds, amphibians, reptiles, or fish. In some aspects, the animal is a human, mouse, C.elegans, rat, fruit fly (Drosophila spp.), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, Japanese rice fish, sea lamprey, pufferfish, tree frog (e.g., Xenopus spp.), monkey, or chimpanzee. The specific cell types contemplated include haploid cells, diploid cells, germ cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, embryonic cells, hematopoietic cells, osteocytes, germ plasm cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells. In some aspects, a plurality of cells from an organism can be used.

[0248] The compositions and methods described herein can be used to edit the genome of an animal cell in various ways. In one aspect, it may be desirable to delete one or more nucleotides. In another aspect, it may be desirable to insert one or more nucleotides. In one aspect, it may be desirable to replace one or more nucleotides. In another aspect, it may be desirable to modify one or more nucleotides via a covalent or non-covalent interaction with another atom or molecule.

[0249] Genome modification can be used to achieve genotypic and / or phenotypic changes in a target organism. Such changes are preferably associated with the improvement of a target phenotype or physiologically important characteristic, the correction of an endogenous defect, or the expression of a certain type of expression marker. In some aspects, the target phenotype or physiologically important characteristic is related to the overall health, adaptability or fertility of the animal, the ecological adaptability of the animal, or the relationship or interaction between the animal and other organisms in the environment.In some aspects, the phenotype of interest or physiologically important characteristic is selected from the group consisting of improved overall health, disease reversal, disease modification, disease stabilization, disease prevention, treatment of parasitic infections, treatment of viral infections, treatment of retroviral infections, treatment of bacterial infections, treatment of neurological disorders (such as but not limited to multiple sclerosis), endogenous genetic defects (such as but not limited to metabolic disorders, rickets, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Barth syndrome, breast cancer, Charcot-Marie-Tooth, colon cancer, Cri du chat, Crohn's disease, cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia Thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome Syndrome, WAGR syndrome, and Wilson Disease, treatment of innate immune disorders (such as, but not limited to, immunoglobulin subclass deficiencies), treatment of acquired immune disorders (such as, but not limited to, AIDS and other HIV-related disorders), treatment of cancer, and treatment of diseases, including rare or "orphan" conditions, for which no effective treatment options are available otherwise.

[0250] Cells genetically modified using the compositions or methods described herein can be transplanted into a subject for purposes such as gene therapy, for example, to treat a disease or as an antiviral, antipathogen or anticancer therapeutic, for production of genetically modified organisms in agriculture, or for biological research.

[0251] Plant cells and plants

[0252] Examples of monocots that can be used include, but are not limited to, corn (maize), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), broomcorn millet (Panicum miliaceum), foxtail millet (Setaria italica), foxtail millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), bananas (Musas spp.), palms, ornamentals, turf grasses, and other grasses.

[0253] 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, 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).

[0254] 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), banana (Musas spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia oleracea), walnut (Canadian walnut ... integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals, and conifers.

[0255] Vegetables that can be used include tomatoes (Lycopersicon esculentum), 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.), hydrangea (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.

[0256] Conifers that can be used include pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis).

[0257] In certain embodiments of the present disclosure, fertile plants are plants that produce viable male and female gametes and are self-fertile. Such self-fertile plants can produce offspring 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 plants that are not self-fertile because the plant does not produce viable or otherwise fertilizable male or female gametes or both.

[0258] The present disclosure can be used for breeding of plants comprising one or more introduced traits or edited genomes.

[0259] A non-limiting example of how two traits can be stacked into a genome at a genetic distance of, for example, 5 cM from each other is described below: A first plant comprising a first transgenic target site integrated into a first DSB target site within a genomic window and without a first genomic locus of interest is crossed with a second transgenic plant, the second transgenic plant comprising a genomic locus of interest at a different genomic insertion site within the genomic window, and the second plant not comprising the first transgenic target site. Approximately 5% of the plant offspring from this cross will have within the genomic window the first transgenic target site integrated into the first DSB target site and the first genomic locus of interest integrated at a different genomic insertion site. Offspring plants with two sites in a defined genomic window can be further crossed with a third transgenic plant, the third transgenic plant comprising within the defined genomic window a second transgenic target site integrated into a second DSB target site, and / or a second genomic locus of interest and lacking the first transgenic target site and the first genomic locus of interest. Offspring with the first transgenic target site, the first genomic locus of interest, and the second genomic locus of interest integrated at different genomic insertion sites within the genomic window are then selected. Such methods can be used to produce plants comprising complex trait loci having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic target sites integrated into the DSB target site and / or a target genomic locus of interest at different sites within a genomic window. In this way, various complex trait loci can be produced.

[0260] 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 particularly incorporated by reference.

[0261] Examples

[0262] Below 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 numerals (e.g., amounts, temperatures, etc.) used, efforts have been made to ensure their accuracy, but some experimental errors and deviations should still be allowed.

[0263] Example 1: Two-step repair of disease susceptibility genes

[0264] Northern leaf blight (NLB), induced by the fungal pathogen Exserohilum turcicum (formerly known as Helminthosporium turcicum), is a serious leaf blight of corn in many tropical and temperate environments. Symptoms can range from cigar-shaped lesions on the lower leaves to complete destruction of the leaves, reducing the amount of leaf surface area available for photosynthesis. The reduction in photosynthetic capacity leads to a deficiency of carbohydrates required for grain filling, which affects grain yield. Tropical mid-altitude areas (approximately 900-1600 m above sea level) have a climate that is particularly favorable for northern leaf blight because the dew period is long and temperatures are moderate. However, in temperate environments (such as in the United States), northern leaf blight may also produce 30%-50% losses during the wet season, especially if infection is established on the upper leaves of the plant during the silking period. The most effective and preferred method of controlling northern leaf blight is to plant resistant hybrids. Certain natural disease resistance maize genes, such as Ht1, Ht2, Ht3, Htm1, Htn1, HtN, HtP, ht4, and rt, can control resistance to specific pathogen species (Welz and Geiger 2000. Plant Breeding. 119(1): 1-14; Ogliari et al. 2005. Genet Mol Biol 28: 435-439; Hurni et al. 2015 PNAS 112(28): 8780-5). However, introgressing resistance genes into other inbred lines is a difficult task, which may or may not result in yield losses due to linkage drag. The limitations of introgressing northern leaf blight resistance into corn lines in conventional breeding can be overcome by editing genes that confer enhanced resistance to northern leaf blight (such as, for example, Ht1 and NLB18), or by moving the resistance alleles of Ht1 and NLB18 to another site in the genome so that enhanced resistance to northern leaf blight can be obtained by introgressing a single genomic locus containing multiple nucleotide sequences (each conferring enhanced resistance to northern leaf blight) into corn plants.

[0265] Chromosome 8 of the maize genome contains multiple NLB alleles, including NLB15, NLB17, and NLB18 ( Figure 2). NLB18 is highly homologous to NLB17, with repetitive sequences near the genome and in other regions of the genome. Here, we describe the generation of CRISPR-Cas corn with improved resistance to NLB, in which the disease-sensitive allele of the NLB18 gene (referred to as NLB18-S) has been replaced with the disease-resistant allele of the NLB18 gene (referred to as NLB18-R). Initially, the technical method of targeted allele replacement was considered to be a single step using a single transformation. However, no plants with allele replacement were recovered in this single transformation method. Plants with a missing NLB18-S allele (referred to as the NLB18-S deletion line) were recovered from the initial transformation. Then a second step was added to the original method. In the second step, the NLB18-R allele was incorporated into the NLB18-S deletion line at the position of the missing NLB18-S allele. Therefore, the resulting line contains the NLB18-R allele at its natural genomic position to replace the NLB18-S allele.

[0266] NLB18-S deletion was achieved by introducing the following two guide RNAs (gRNAs) to generate two cuts in the DNA of the maize inbred line PH1V5T in predetermined positions: one 5' guide RNA homologous to the upstream sequence located in the promoter region of the susceptible allele, and one 3' guide RNA homologous to the downstream sequence located in the 3' UTR of the susceptible allele. Seven plasmids were used to bombard immature maize embryos. Plasmid 1 is a donor of Cas9 endonuclease; plasmids 2 and 3 are donors of 5' guide RNA and 3' guide RNA, respectively; plasmid 4 carries the NLB18-R DNA sequence from the maize inbred line PH26N (which is not present in the product of step 1); plasmids 5 and 6 are donors of two auxiliary genes (zm-odp2 and zm-wus2) for improving embryo response and plant generation frequency; and plasmid 7 is a donor of the nptII selectable marker.

[0267] Although a plasmid donor containing a DNA repair template of the DNA sequence of NLB18-R was included in the transformation; no T0 plants containing the complete NLB18-R DNA sequence were recovered. However, T0 plants with a deletion of the NLB18-S DNA sequence between the two DSBs were recovered. The progeny from these NLB18-S deletion plants were moved to step 2.

[0268] The goal of NLB18-R allele replacement was achieved in a second transformation that introduced a single gRNA to generate a DSB in the DNA sequence at the repair site of the NLB18-S deletion from step 1. Immature maize embryos were subjected to Agrobacterium-mediated transformation with plasmid 8 ( Figure 3). Plasmid 8 is a donor of Cas9 endonuclease; guide RNA; NLB18-R DNA sequence; two auxiliary genes (zm-odp2 and zm-wus) for improving embryo response and plant generation frequency; and nptII selectable marker.

[0269] T0 plants were recovered that contained the complete NLB18-R DNA sequence in the position of the previously deleted NLB18-S. Thus, the product of the two-step process was identical to the one-step process described for the hypothetical product (CRISPR-Cas corn with improved resistance to NLB), where:

[0270] Resistance alleles are alternative alleles of the same corn gene,

[0271] ·Resistant alleles replace sensitive alleles,

[0272] The resistance allele is present in its natural genomic location, and

[0273] Similar results can be found in nature and can be achieved through conventional breeding.

[0274] The products of the two-step approach were molecularly characterized to confirm the absence of NLB18 allelic replacement and integration of unintended DNA sequences from the transformation plasmid, as well as phenotypic confirmation of increased NLB resistance.

[0275] T0 seedlings were analyzed for perfect gene / sequence deletion and random integration of T-DNA into the genome. The results are shown in Table 1.

[0276] Table 1: T0 embryos from Agrobacterium-mediated transformation

[0277]

[0278]

[0279] Allelic replacement was confirmed in T1 plants by capture sequencing (Southern-by-Sequencing), as shown in Table 2. The results are also depicted in Figure 5 Molecular characterization of CRISPR-Cas maize with improved resistance to NLB included analysis to confirm the absence of unintended plasmid DNA from all transformed plasmids. TM, an efficient sequencing-based molecular characterization tool) was used for this analysis (Brink et al., 2019; Zastrow-Hayes et al., 2015). The SbS analysis covers the sequences of all eight plasmids and detects unique junctions created between plant genomic DNA and the unexpected sequence derived from the transformed plasmid if an unexpected plasmid DNA integration has occurred. Plants in which no unexpected plasmid-derived DNA was detected were selected and improved for further development.

[0280] Table 2: T1 embryos from Agrobacterium-mediated transformation

[0281]

[0282] This approach allows gene replacement in one generation, compared to the two-generation method currently used. It also allows simplification, and saves labor and time.

[0283] Example 2: Agrobacterium-mediated transformation and particle bombardment transformation are effective

[0284] Using similar vectors, T0 plants were transformed using particle gun bombardment. As shown in Table 3 (all false positives removed), both methods successfully replaced the susceptible NLB18 allele with the disease resistance allele.

[0285] Table 3: Agrobacterium- and particle bombardment-mediated transformation results

[0286]

[0287] Example 3: Development of Elite Inbred Maize Lines with Disease Resistance

[0288] Figure 3 Describe the general schematic diagram of inbred development.Immature embryos of corn inbred lines were transformed by particle bombardment with the plasmid described above.At the NLB18 locus, T0 plants were analyzed by connecting PCR and Sanger sequencing to identify the plants that NLB18-S was replaced by NLB18-R targeting.The T0 plants that replaced the NLB18-S allele with a complete NLB18-R allele were not identified.The T0 plants (wherein the NLB18-S allele was missing between two DSBs, without any addition or disappearance of extra nucleotides) that confirmed the NLB18-S disappearance were identified, and backcrossed with the wild-type inbred line.The resulting BCO (F1) plants were analyzed based on next generation sequencing (NGS) to confirm the NLB18-S disappearance under the nucleotide level.The BCO (F1) plants that confirmed the NLB18S disappearance were self-pollinated, and the resulting BCO (F2) plants that were identified as being homozygous for the NLB18 S disappearance were self-pollinated. The resulting BCO(F3) immature embryos were used as transformation tissue in step 2.

[0289] like Figure 3 As shown, the BC0 (F3) immature embryos generated in step 1 were transformed with plasmid 8 via an Agrobacterium tumefaciens-mediated method. T0 plants were analyzed by ligation PCR and Sanger sequencing at the NLB18 locus to identify plants with NLB18-R in the targeted position of the previously deleted NLB18-S. Several T0 plants confirmed to contain NLB18-R in the targeted position were backcrossed with a wild-type inbred line. The resulting BC0 (F1) plants were subjected to comprehensive molecular analysis, including: NGS-based analysis to confirm the presence of a single intact NLB18 R at the targeted position: and NGS-based analysis to verify the absence of unexpected plasmid DNA from eight transformation plasmids (seven from step 1 and one from step 2).

[0290] Individual BCO (F1) plants confirmed to contain NLB18-R in the targeted position and without unintentional integrated plasmid DNA were self-pollinated. The resulting BCO (F2) plants were phenotyped for increased NLB resistance. In addition, individual BCO (F1) plants confirmed to contain NLB18R in the targeted position and without unintentional integrated plasmid DNA were backcrossed with wild-type inbred lines for superior inbred line development.

[0291] Example 4: Confirmation of allele replacement in inbred lines

[0292] Next generation sequencing (NGS) based techniques were used to characterize the replacement of NLB18-S with NLB18-R by comparing the sequence of the NLB18 gene region of wild type corn containing intact NLB18-S with the same region in BC0 (F1) plants from step 2. The expected change in CRISPR-Cas corn with improved resistance to NLB is to replace NLB18-S with the NLB18-R gene sequence between the two homology arms. Sequencing analysis showed that a single intact NLB18-R allele was present at the location of the NLB18-S deletion and no DNA sequence changes were detected through the homology arm region, where the natural homology directed repair (HDR) mechanism was able to allow allele replacement without creating novel genetic material combinations.

[0293] Example 5: Confirmation of the absence of unintended DNA integration from the transformation plasmid in the inbred line

[0294] Individual BCO (F1) plants were analyzed using SbS. Several genetic elements on the plasmid used for transformation are endogenous to maize ( Figure 2 and Figure 3). It is expected that SbS analysis of these sequences will detect these endogenous sequences in CRISPR-Cas maize with improved resistance to NLB in their natural environment. By examining the junction sequence between the plasmid sequence and the maize genomic sequence, it is apparent that there are unintended plasmid-derived sequences in the CRISPR Cas maize with improved resistance to NLB.

[0295] Thus, it was confirmed that the plasmid 1 sequence was absent because no unique plasmid genome junctions were detected, and only the expected maize endogenous genetic elements were detected in the natural genomic environment in the CRISPR-Cas maize with improved resistance to NLB genomic DNA. Similar analyses were performed on the remaining seven plasmids used in the two transformation steps for generating CRISPR-Cas maize with improved resistance to NLB. It was also confirmed that the plasmid 2, 3, 4, 5, 6, 7, and 8 sequences were absent because no unique plasmid genome junctions were detected, and only the expected endogenous elements in their natural genomic environment were detected in the BC0 (F1) plants advanced for the development of CRISPR-Cas lines with improved resistance to NLB in inbred maize.

[0296] Therefore, SbS analysis, which provides sequence-level information, was used to confirm the absence of unintended plasmid DNA from the eight plasmids used to generate CRISPR-Cas maize with improved resistance to NLB without creating novel combinations of genetic material. Plants in which unintended plasmid-derived DNA was not detected were then improved for further development.

[0297] Example 6: Confirmation of increased resistance to NLB

[0298] The expected phenotype of CRISPR-Cas NLB corn is improved resistance to NLB. Resistance to NLB can be confirmed by assessment based on visual inspection of plants after inoculation with the fungus that causes NLB. Improved resistance to NLB was confirmed for inbred lines with NLB18-R compared to wild-type inbred lines with NLB18-S.

[0299] Multiple BC0 (F2) plants were grown in a greenhouse under standard conditions. When the plants reached the V3-V4 stage, i.e., the stage at which the root collars of the third and fourth leaves, respectively, were visible (Abendroth et al., 2011), they were inoculated with a conidial suspension of Setosphaeria turcica. The conidia were suspended in sterile distilled water to a density of 10,000 spores / mL. The suspension was applied to the whorls of each plant at 100 μL / plant. 14 days after inoculation, the plants were scored as resistant or susceptible based on visual observation of characteristic symptoms of NLB (Munkvold, 2016). Plants susceptible or resistant to NLB, respectively, were easily identified by the presence or absence of typical lesions ( Figure 4 ).

[0300] These results confirmed that targeted allelic replacement using CRISPR-Cas technology to replace NLB18-S with NLB18-R resulted in the expected phenotypic change of increased resistance to NLB.

[0301] Although the examples herein describe replacing an endogenous gene with a heterologous polynucleotide to achieve a phenotypic change, one skilled in the art will appreciate that any endogenous polynucleotide (such as, but not limited to, a regulatory element, DNA encoding RNA, etc.) can be replaced by the methods provided herein.

Claims

1. A method for replacing an unwanted polynucleotide in the genome of an organism, wherein include: (a) providing to cells of the organism: (i) Cas endonucleases, and (ii) a first and a second guide RNA, wherein each of the first and the second guide RNA is capable of hybridizing to a first and a second target sequence in the genome of the organism, respectively; wherein the first and the second target sequences in the genome are located flanking the unwanted polynucleotide; wherein the Cas endonuclease forms a complex with the first guide RNA to create a break at or near the first target sequence in the genome of the organism, wherein the Cas endonuclease forms a complex with the second guide RNA to create a break at or near the second target sequence in the genome of the organism, wherein the unwanted polynucleotide is deleted from the genome, and wherein the first and second target sequences are brought into closer proximity to each other, thereby forming a third target sequence; (b) providing to cells of the organism: (i) a heterologous polynucleotide that shares at least 75% identity with the undesired polynucleotide, wherein the heterologous polynucleotide is flanked by sequences that are identical to sequences that flank the third target sequence; (ii) a third guide RNA capable of hybridizing to the third target sequence; wherein the Cas endonuclease forms a complex with the third guide RNA to create a break at or near the third target sequence in the genome of the organism, wherein the heterologous polynucleotide is inserted into the genome of the organism, Wherein the method is for non-therapeutic purposes.

2. A method for modifying a polynucleotide in the genome of an organism, wherein the polynucleotide is located in a region of a repetitive sequence, the method comprising sequentially introducing into at least one cell of the organism: (a) a Cas endonuclease, and a first and a second guide RNA, wherein each of the first and the second guide RNA is capable of hybridizing to a first and a second target sequence in the genome of the organism, respectively; wherein the first and the second target sequences in the genome are located at the sides of the unwanted polynucleotides; wherein the Cas endonuclease forms a complex with the first guide RNA to create a break at or near the first target sequence in the genome of the organism, wherein the Cas endonuclease forms a complex with the second guide RNA to create a break at or near the second target sequence in the genome of the organism, wherein the unwanted polynucleotide is deleted from the genome, and wherein the first and second target sequences are brought into closer proximity to each other, thereby forming a third target sequence; (b) a heterologous polynucleotide that shares at least 75% identity with the undesired polynucleotide, wherein the heterologous polynucleotide is flanked by sequences identical to sequences flanking the third target sequence; and a third guide RNA that is capable of hybridizing to the third target sequence; wherein the Cas endonuclease forms a complex with the third guide RNA to create a break at or near the third target sequence in the genome of the organism, wherein the heterologous polynucleotide is inserted into the genome of the organism, wherein the method is for non-therapeutic purposes.

3. A method of producing an organism with an improved phenotype, comprising sequentially introducing into at least one cell of the organism: (a) a Cas endonuclease, and a first and a second guide RNA, wherein each of the first and the second guide RNA is capable of hybridizing to a first and a second target sequence in the genome of the organism, respectively; wherein the first and the second target sequences in the genome are located at the sides of the unwanted polynucleotides; wherein the Cas endonuclease forms a complex with the first guide RNA to create a break at or near the first target sequence in the genome of the organism, wherein the Cas endonuclease forms a complex with the second guide RNA to create a break at or near the second target sequence in the genome of the organism, wherein the unwanted polynucleotide is deleted from the genome, and wherein the first and second target sequences are brought into closer proximity to each other, thereby forming a third target sequence; (b) a heterologous polynucleotide that shares at least 75% identity with the undesired polynucleotide, wherein the heterologous polynucleotide is flanked by sequences identical to sequences flanking the third target sequence; and a third guide RNA that is capable of hybridizing to the third target sequence; wherein the Cas endonuclease forms a complex with the third guide RNA to create a break at or near the third target sequence in the genome of the organism, wherein the heterologous polynucleotide is inserted into the genome of the organism, wherein the method is for non-therapeutic purposes.

4. The method of claim 1 or claim 2 or claim 3, further comprising introducing a second Cas endonuclease into step (b).

5. The method of claim 1 or claim 2 or claim 3, wherein the cell in (b) is from a descendant derived from the cell in (a).

6. The method of claim 1 or claim 2 or claim 3, wherein the genome of the organism comprises a highly repetitive region.

7. The method of claim 1 or claim 2 or claim 3, wherein the unwanted polynucleotides comprise genes that impart a deleterious phenotype to the organism.

8. The method of claim 1 or claim 2 or claim 3, wherein the organism is a plant.

9. The method of claim 1 or claim 2 or claim 3, wherein the organism is corn.

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