Invented by Yajie Niu, Randall William Shultz, Maria Margarita D. Unson, Michael Andreas KOCK, John P. Casey, Jr., Inari Agriculture Inc

The market for methods for creating multiple targeted insertions within a cell of a plant is rapidly growing as advancements in genetic engineering continue to revolutionize the agricultural industry. This innovative technique allows scientists to introduce multiple desired traits into a plant’s genome simultaneously, resulting in improved crop yields, enhanced nutritional content, and increased resistance to pests and diseases. Traditionally, plant breeding involved cross-pollination and selective breeding to achieve desired traits in crops. However, this process was time-consuming and often resulted in unpredictable outcomes. With the advent of genetic engineering, scientists can now directly manipulate a plant’s DNA to introduce specific genes that confer desired traits. The method for creating multiple targeted insertions within a cell of a plant involves the use of molecular tools such as CRISPR-Cas9, TALENs, or zinc finger nucleases. These tools act as molecular scissors, allowing scientists to precisely cut and edit the plant’s DNA at specific locations. By introducing multiple desired genes simultaneously, scientists can create plants with multiple beneficial traits in a single generation. One of the key advantages of this method is its ability to accelerate the breeding process. Traditional breeding methods can take several generations to achieve the desired traits, while the targeted insertion method allows scientists to introduce multiple traits within a single generation. This significantly reduces the time required to develop new crop varieties, enabling farmers to access improved crops more quickly. Another advantage of this method is its precision. By targeting specific locations in the plant’s genome, scientists can ensure that the desired traits are inserted at the intended sites. This precision minimizes the risk of unintended genetic changes and increases the efficiency of the genetic modification process. The market for methods for creating multiple targeted insertions within a cell of a plant is driven by the increasing demand for sustainable and high-yielding crops. As the global population continues to grow, there is a pressing need to produce more food using limited resources. By introducing multiple beneficial traits into crops, scientists can develop varieties that are more resilient to environmental stresses, require fewer inputs such as water and pesticides, and produce higher yields. Furthermore, this technology offers significant potential for improving the nutritional content of crops. By introducing genes that enhance the production of vitamins, minerals, or other beneficial compounds, scientists can develop crops with improved nutritional profiles. This has the potential to address malnutrition and improve public health, particularly in developing countries where access to nutritious food is limited. The market for methods for creating multiple targeted insertions within a cell of a plant is also driven by the increasing awareness of the environmental impact of conventional farming practices. By developing crops with enhanced resistance to pests and diseases, farmers can reduce their reliance on chemical pesticides, leading to a more sustainable and environmentally friendly agriculture. However, despite the numerous benefits, there are also concerns surrounding the use of genetic engineering in agriculture. Critics argue that the long-term effects of genetically modified crops on human health and the environment are still unknown. Additionally, there are ethical considerations regarding the patenting of genetically modified organisms and the potential for corporate control over the global food supply. In conclusion, the market for methods for creating multiple targeted insertions within a cell of a plant is expanding rapidly due to the potential to develop crops with improved traits and increased yields. This technology offers significant benefits in terms of sustainability, nutritional content, and resistance to pests and diseases. However, it is important to address the ethical and environmental concerns associated with genetic engineering to ensure its responsible and safe use in agriculture.

The Inari Agriculture Inc invention works as follows

The invention is a novel plant, seed, composition, and method for improving plant breeding.

Background for Method for creating multiple targeted insertions within a cell of a plant

Plant breeding and engineering relies primarily on Mendelian or recombinant genetics. Wang et. al., Nature Biotechnology 32(9) (2014) describe TALEN-mediated edits (indels), of wheat MLO gene. This method involves transforming wheat protoplasts using a TALEN plasmid, a plasmid containing a selectable mark (bar), and then selecting herbicide-resistant calli. Transgenic seedlings are then generated. Wang describes the knock-in of GFP-coding sequences at TaMLO sites in wheat protoplasts using non-homologous ends joining (NHEJ). The method involves transformation of wheat protoplasts using T-MLO and GFP donor plasmids.

Zhang et al., Nat. Commun. 7:12617, (2016) describes a way to introduce a targeted indel within a wheat locus. The method involves the use of plasmids that encode or express CRISPR/Cas9 and guide RNA, and the insertion into wheat callus cell.

Liang, Z. et al., Nat. Commun. 8:14261 (2017) describes a technique for introducing targeted mutations into homologs (TaGW2) of a wheat gene. The method involves delivering Cas9/sgRNA-RNPs to embryonic wheat calli by particle bombardment or PEG mediated transformation.

PCT application No. WO2016007948 published on Jan. 14, 2016 and entitled?Agronomic traits modification using guideRNA/cas endonuclease system and methods of usage,? The methods described are for introducing specific modifications to the maize gene. The methods involve homologous hybridization and/or transformation of maize eggs with plasmids and/or selectable markers.

Disclosed are methods to provide novel plant cells, or plant protoplasts. They also include plant callus, tissues, parts, or whole plants. And seeds with one or more altered sequences of genetic material. The methods and compositions described in this patent allow for the stacking or combining of alleles, without introducing unwanted epigenetic or genetic variation into the modified cells or plants. These targeted modification methods have a much higher efficiency and reliability than traditional plant breeding. They can be used to supplement traditional breeding techniques, but they can also replace them.

The invention discloses methods of providing novel plant cells, plant protoplasts or callus, tissues or other parts, whole plants and seeds with one or more altered gene sequences.

The The Some These In A As In In In In In This The Embodiments include use of the method to integrate or introduce into a genome sequence of a promoter or promoter-like element (e. g., sequence of an auxin-binding or hormone-binding or transcription-factor-binding element, or sequence of or encoding an aptamer or riboswitch), or a sequence-specific binding or cleavage site sequence (e. g., sequence of or encoding an endonuclease cleavage site, a small RNA recognition site, a recombinase site, a splice site, or a transposon recognition site). In embodiments, the method is used to delete or otherwise modify to make non-functional an endogenous functional sequence, such as a hormone- or transcription-factor-binding element, or a small RNA or recombinase or transposon recognition site. In The In another example, the method is used to integrate a polynucleotide-encoded heterologous small RNA recognition site sequence at a DSB in a sequence of interest in a genome, wherein when the small RNA is present (e. g., expressed endogenously or transiently or transgenically), the small RNA binds to and cleaves the transcript of the sequence of interest that contains the integrated small RNA recognition site. In another example, the method is used to integrate in the genome of a plant or plant cell a polynucleotide-encoded promoter or promoter-like element that is responsive to a specific molecule (e. g., an auxin, a hormone, a drug, an herbicide, or a polypeptide), wherein a specific level of expression of the sequence of interest is obtained by providing the corresponding specific molecule to the plant or plant cell; in a non-limiting example, an auxin-binding element is integrated into the promoter region of a protein-coding sequence in the genome of a plant or plant cell, whereby the expression of the protein is upregulated when the corresponding auxin is exogenously provided to the plant or plant cell (e. g., by adding the auxin to the medium of the plant cell or by spraying the auxin onto the plant). A The

The invention also provides a composition that includes a plant-cell and a donor polynucleotide molecule (such as double-stranded or single-stranded stranded, DNA/RNA, hybrids, etc.) capable of being (or having) its sequence integrated into a double strand break in genomic sequencing in the plant-cell. In different embodiments, the cell can be an isolated protoplast or plant cell, or it could be in a monocot or dicot, a zygotic embryo or somatic embryo or seed, plant part or tissue. In some embodiments, the plant cell can be divided or differentiated. In some embodiments, the plant cell can be haploid, diploic, or polyploid. In embodiments the genome of the plant cell has a double-stranded break (DSB), at which DSB the polynucleotide donation molecule is integrated. In embodiments, a DSB in the genome of the plant cell is induced by including a DSB inducing agent in the composition, such as a DSB inducing endonuclease (e. g. RNA guided nucleases, such as a Cas9 or a CasX), guide RNAs which direct cleavage to an RNA-guided Nuclease. The dsDNA molecule then is integrated using the Compositions that include a plant cell and atleast one dsDNA are disclosed. The compositions also contain at least a ribonucleoprotein containing a nuclease site-specific (e.g. Cas9 or Cpf1, CasX or CasY or C2c1, C2c3), as well as at least a guide RNA. In some embodiments, the polynucleotide molecule can be double-stranded RNA or DNA, or a mixture of both, with a blunt end, terminal overhangs or chemical modifications, such as phosphorothioate or detectable labels. In other embodiments the polynucleotide molecule can be a single-stranded mononucleotide made of DNA, RNA, or a mixture of DNA and RNA. It can also be chemically altered or labeled. The polynucleotide molecule can be a single-stranded polynucleotide composed of DNA or RNA, or a combination of both. It may also include a nucleotide that has a specific function when it is integrated at the DSB site. In various non-limiting examples, the polynucleotide donation molecule can include: a sequence that can be recognized by a binding agent, that binds a molecule in particular, that encodes a molecule of RNA or an amino-acid sequence, that responds to a change in physical environment, that encodes a molecule of RNA or an amino-acid sequence responsive to a change in physical environment, that encodes a molecule of RNA or an amino

The invention also provides a reaction mix that includes: (a), a plant cell with at least one double strand break (DSB), at a particular locus of its genome, (b), a polynucleotide molecule (such as a DNA or RNA hybrid or single-stranded nucleotide) capable of being (or having its sequence) integrated at the DSB. This polynucleotide molecule can have In some embodiments, the plant cell can be an isolated cell or a plant protoplast. In different embodiments, the cell can be an isolated cell, a plant protoplast or a part of a monocot or dicot, a zygotic embryo or a somatic embryo or a seed, a plant part or a plant tissue. In some embodiments, the plant cell can be divided or differentiated. In some embodiments, the plant cell can be haploid, diploic, or polyploic. In some embodiments, the polynucleotide donation molecule has a nucleotide that is useful when it is integrated at the site of DSB. In various non-limiting examples, the polynucleotide molecule can include: a sequence that can be recognized by a binding agent, or that binds a molecule in a particular way, or encodes a molecule in a certain way, or is responsive or encodes a molecule in a certain way, or is heterologous, or has secondary structure, such as double-stranded stems, stem-loops, In embodiments in which the donor sequence is inserted via NHEJ the donor molecule does not contain a sequence of nucleotides that are homologous or complementary to a sequence of nucleotides flanking a DSB. In the embodiments described, the donor polynucleotide does not contain a nucleotide that is identical to a T-DNA border of Agrobacterium. The methods allow for the precise insertion at two or multiple loci of a heterologous non-homologous polynucleotide donor sequence (e.g., a non-coding sequence such as a regulator or expression-modifying component) according to embodiments. In some embodiments, one or more of these insertions and targeted modifications modify the expression of a gene or sequence that is of interest. In some embodiments, two or more loci are located in different genes or sequences. In embodiments, two or more loci in a genome are alleles. When all alleles are modified the same way for a gene or sequence, it results in homozygous modifications of the gene. “For example, embodiments allow targeted modification to both alleles within a given gene or sequence of interest in a plant with a 2n ploidy (n=1), or all three alleles within a plant with a 2n ploidy (n=1.5), or all six alleles for a gene inside a plant that is a hexaploid or a 2n ploidy (2n = 3).

In a second aspect, the invention provides polynucleotides for disrupting gene transcription, wherein each polynucleotide has at least 11 nucleotides and is either double-stranded, containing at least 18 base pairs, and encoding a stop codon on each of the possible reading frames on each strand. In some embodiments, the molecule is double-stranded (dsDNA), or a hybrid of double-stranded RNA and DNA. It includes at least 18 base pairs and encodes at least one stop code in each reading frame possible on either strand. In embodiments, a polynucleotide can be a single stranded nucleotide or a DNA/RNA hybrid including at least 11 nucleotides. The stop codon is encoded in every possible reading frame. This polynucleotide can be used in the methods described herein where, after a sequence encoded in the polynucleotide has been integrated or inserted in a genome, at the site of a DSB, the sequence from the heterologously inserted sequence serves to stop the translation of the transcription containing both the sequence and the heterologously inserted sequence. Polynucleotide embodiments include those in which the polynucleotide contains one or more chemical labels or modifications, such as at least one phosphorothioate label.

The The The

The The The

The invention also provides a method for modifying a cell of a flowering plant by creating a plurality targeted modifications. This method involves contacting the genome using one or multiple targeting agents, which contain or encode nucleic acids or peptides that bind to or near specific target sites in the plant’s genome (for example, nucleases or polynucleotides encoding nucleases or guide RNAs), and the method does not require a separate step to identify an individual modification ?Lacks homology? In the context of targeted modification described herein, lack of homology means that the donor’s sequence does not have enough complementarity or homology to allow it to bind with the genomic sequences flanking the site where the genomic insertion is to be made. The methods allow for precise insertions of at least one heterologous non-homologous polynucleotide sequence at two or more predetermined sites in the genome. The donor sequence inserted can be either coding (e.g. protein or RNA-coding) sequence, non-coding (e.g. regulatory elements) or even a combination. The sequence encoded by a polynucleotide is heterologous in relation to the genomic locus where the sequence is inserted. In some embodiments, the insertion of non-coding sequences modifies expression levels of endogenous genes located in cis (e.g. 5? In embodiments, insertion of a non-coding donor sequence modifies expression of an endogenous gene located cis to (e.g., 5? To) the inserted code. In one embodiment, the polynucleotide donors used in the method are a single-stranded DNA, single-stranded RNA, single-stranded hybrid DNA-RNA, or duplex RNA/DNA. In a related embodiment, the modified plant cells of the method are meristematic cells, embryonic or germline cells. In another embodiment, the methods described herein, when repeated or applied to a group of cells, produce an efficiency of atleast 1%. In another embodiment, a targeted plant cell with a ploidy value of 2n is used. n can be any of the following values: 0.5, 1, 1,5, 2, 2.5, 3.5, 4, 5, or 6. The method then generates 2n modifications at 2n sites of the predetermined targets within the plant genome.

The invention also provides a method for modifying a cell of a flowering plant by creating a plurality targeted modifications. This is done by contacting the genome using one or multiple targeting agents. These agents contain or encode predetermined nucleic acids or peptides that bind to or near specific target sites in the plant’s genome. In one embodiment, the polynucleotide donors used in the method do not have homology with the genome sequences near the site of insertion. In a related embodiment, in which the modified plant cells are meristematic, embryonic, or germline cells, they can be referred to as a related embodiment. In another embodiment, the repetition of the described methods results in an efficiency of atleast 1%. In another embodiment, a targeted plant cell is 2n ploidy, where n is a selected value from the following: 0.5, 1, 1,5, 2, 2.5. 3, 3.5. 4, 5, 6, and 7, where the method creates 2n modified sequences at 2n loci within the genome of the plant cell.

The invention also provides a method for modifying a cell of a flowering plant by creating a plurality targeted modifications. This is done by contacting the genome using one or multiple targeting agents. These agents contain or encode predetermined nucleic acids or peptides that bind to or near target sites in the plant’s genome. In another embodiment, one or more polynucleotide donors encode a predetermined sequence that is then inserted into the genome of the plant cell. The polynucleotide donors can be a DNA, RNA, DNA-RNA hybrid, or duplex RNA/DNA molecule. In another embodiment, one or more polynucleotide donors do not have homology with the genome sequences near the site of insertion. In another embodiment, the repetition of the method leads to an efficiency of atleast 1%. This can be e.g. atleast 2%, or atleast 5%, or atleast 7%, or atleast 10%, 15%, 25%, 30%, and more. The efficiency is calculated by dividing the total number cells targeted by the number of successfully target cells. In another embodiment, the target plant cell has a 2n ploidy, where n is a selected value from the following: 0.5, 1, 1,5, 2, 2.5, 3.5, 4, 5, 6, and 7, wherein the method creates 2n modified sequences at 2n loci within the genome of the plant cell.

The invention also provides a method for modifying a cell of a flowering plant by creating a plurality targeted modifications. This is done by contacting the genome using one or multiple targeting agents. These agents contain or encode predetermined nucleic acids or peptides that In one embodiment, the modified cell is an embryonic, germline, or meristematic plant cell. In another embodiment, one of the targeted modification is the insertion of a sequence encoded by a polynucleotide molecule. At least one polynucleotide molecule can be a DNA single-stranded, an RNA single- In a related embodiment, one or more polynucleotide donors used in the methods do not have homology with the genome sequences near the site of insert. In another embodiment, the target plant cell has a 2n ploidy, where n is a selected value from the following: 0.5, 1, 1,5, 2, 2.5, 3.5, 4, 5, and 6. The method produces 2n targeted modifications in 2n loci within the genome

In certain embodiments, two of the targeted modifications are insertions between 10 and 300 nucleotides, between 3 and 400, between 10 to 350, between 18 to 350, between 18 to 200, or between 10 and 150 nucleotides. In some embodiments, the two targeted modifications are insertions of between 10 to 350 nucleotides, between 18 to 350 nucleotides, between 18-200 nucleotides, between 10 and150 nucleotides, or between 11 and100 nucleotides.

In a second variation of the above described methods, at least two inserts are made and at least one insertion is an upregulatory segment. In a third variation, the methods of targeted modification described above create or insert at least one transcription-factor binding site. In a further variation of the above described methods, the insertion of predetermined sequences in the plant genome is accompanied by deletions of sequences.

In a further embodiment, the above-described targeted modification methods include obtaining and breeding a plant from a modified plant cell. In a further embodiment, the described methods include a step that introduces additional epigenetic or genetic changes to the modified cell or plant grown from it.

In an embodiment of the methods of targeted modification described above, two or more targeted insertions up- or lower-regulate expression independently. A targeted insertion can increase expression by at least 10%, 15-folds, 20-folds, 30-folds, 40-folds, 50-folds, or 100-folds. In some embodiments expression can be increased by 10-100%, between 2-fold and 5 fold; between 2 and 10 fold; between 10-fold to 50-fold or between 100-fold to 1000-fold. In some embodiments, the expression may be decreased by at least 1%.

In a related embodiment, the invention provides a composition for targeting a genome comprising a donor polynucleotide tethered by a cRNA using a covalent, non-covalent, or combination of both covalent and noncovalent bonds. The invention also provides a composition to target a genome that comprises a donor polynucleotide attached to a cRNA via a covalent, non-covalent, or combination of both covalent and noncovalent bonds.

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