Patent Grant
11608506
This document relates to materials and methods for inducing genetic alterations in meristematic plant tissue.
The ability to generate plants with a desired genetic makeup, whether commercially or for basic research, is limited by two facets—the delivery of genetic engineering (GE) reagents and the subsequent generation of edited tissues. Procedures for delivery of GE reagents typically utilize the gene transferring bacterium Agrobacterium tumefaciens, or physical means such as particle bombardment. After delivery of reagents, many protocols require subsequent regeneration of the edited somatic tissue into whole plants. Transformed somatic tissue often is pushed to de-differentiate (into callus) and then re-differentiate using tissue culture media containing specific hormone ratios required to drive shoot or root formation. Tissue culture techniques can be complicated by the fact that a majority of agriculturally relevant crop lines are recalcitrant to tissue culture regeneration. Additionally, this process can be time consuming and technically demanding, requires sterile conditions, and may be subject to undesired genetic alterations due to prolonged exposure to hormones. Efforts also may be confounded because resulting tissues can present non-specific chimerism for the transgene, requiring more than one generation to fix the genetic event of interest. All of these limitations render current practices for plant genetic engineering non-ideal for large scale generation of edited plants in a high throughput manner.
Developmental regulatory genes have been employed to generate tissues with modifications of interest. For example, controlled expression of the maize regulatory genes WUSCHEL (WUS) and BABY BOOM (BBM) induced somatic embryogenesis from immature maize embryos, as well as other somatic tissues (Lowe et al., Plant Cell 28(9):1998-2015, 2016; and Mookkan et al., Plant Cell Reports 36(9): 1477-1491, 2017). Hundreds of plantlets were recovered from these somatic embryos even in lines previously recalcitrant to tissue culture, and the generated somatic embryos were capable of developing into full plants with the transgene cassette of interest. Despite this improvement to standard monocot regeneration practices, however, the generation of full plants still required tissue culture steps, sterile technique, and explant handling. In addition, these techniques were only demonstrated in monocot species and have not been implemented in any dicot species.
This document provides new techniques that avoid the constraints noted above and provide a boon for both basic research and commercial germplasm production. The combination of developmental regulators WUS and SHOOT MERISTEMLESS (STM), as well as other regulator combinations, can have an impact on patterning and formation of shoot meristems (Gallois et al., Development 129:3207-3217, 2002), and these developmental regulators have been ectopically expressed in Arabidopsis thaliana and other species to create meristem-like tissue. The methods described herein are based, at least in part, on the discovery that such genes can be combined with GE reagents to promote the formation of edited meristematic tissue that can flower and produce seed. The resultant seed is derived from a single meristematic cell, and therefore represents a clonal genetic editing event that provides an abundance of edited seed after one generation. The direct delivery method described herein also provides the advantage of avoiding tissue culture, which reduces the time needed to regenerate tissues and considerably simplifies the process of generating GE events. Thus, the methods described herein can circumvent the limitations of current plant regeneration protocols, and greatly enhance the potential for development of GE plant lines for both commercial use and basic research.
Thus, this document is based, at least in part, on the development of methods for delivery of developmental regulators to whole plants to induce the transdifferentiation of somatic plant cells in vivo for the production of meristems. These meristems can carry transgenic insertions or genetic editing events to the next generation, creating seed with a GE event of interest in a fraction of the time needed using current standard protocols. The techniques described herein can simplify protocols for transformation, remove requirements for tissue culture, and be accessible to labs with diverse skill sets.
In a first aspect, this document features a method for generating plant tissue having one or more genetic modifications of interest. The method can include (a) introducing into plant cells (i) nucleic acid encoding one or more developmental regulators that, when expressed in the plant cells, induce meristem formation from the plant cells, and (ii) nucleic acid comprising one or more sequences that, when expressed, modify a plant cell to achieve one or more genetic modifications of interest; and (b) deriving de novo tissue from plant cells identified as having the one or more genetic modifications of interest. The one or more developmental regulators can include one or more of Baby Boom, Isopentenyl Transferase, Irrepressible Variants of Monopteros, Shoot Meristemless, and Wuschel. The introducing can be by Agrobacterium, and the nucleic acid encoding one or more developmental regulators and the nucleic acid comprising one or more sequences that modify a plant cell can be included on the same T-DNA or on separate T-DNAs. The method can include introducing nucleic acid encoding two or more developmental regulators into the plant cells by Agrobacterium, where the two or more developmental regulators are encoded by a single T-DNA or are encoded by separate T-DNAs. The method can include introducing nucleic acid encoding two or more developmental regulators into the plant cells by Agrobacterium, where the two or more developmental regulators are encoded by two or more strains of Agrobacterium. The introducing can include electroporation, biolistics, particle bombardment, chemical transfection, nanoparticle delivery, or viral infection. The introducing can include transient transformation or stable transgenesis. The plant cells into which the nucleic acids are introduced can be within a differentiated tissue, within an undifferentiated tissue, within a whole plant, within a germinating seedling, or within a plant part taken from a plant. The plant cells can be cells of a monocotyledonous plant, or cells of a dicotyledonous plant. The one or more sequences that modify a plant cell can include a transgene that, when expressed in the plant cells, achieves an agriculturally relevant trait (e.g., herbicide tolerance). The one or more sequences that modify a plant cell can include a transgene that, when expressed, edits the plant DNA. For example, the one or more sequences that modify a plant cell can include a nucleotide sequence encoding a targeted endonuclease (e.g., a meganuclease, zinc finger nuclease, transcription activator-like effector nuclease, or Clustered Regularly-Interspaced Short Palindromic Repeats-associated nuclease). The one or more sequences that modify a plant cell can encode a targeted enzyme that modifies plant DNA (e.g., a cytosine deaminase or an adenosine deaminase, such as BE3 or ABE). The one or more sequences that modify a plant cell can encode a targeted endonuclease and can include a repair template to introduce one or more specific modifications into the plant genome. The de novo tissue can be meristematic and can be capable of deriving new tissue carrying the one or more genetic modifications of interest. The new tissue can include a branch, a flower, or a root.
In some cases, the method can include (a) using Agrobacterium, introducing into cells of a germinating seedling or a portion thereof nucleic acid encoding the one or more developmental regulators, wherein expression of the one or more developmental regulators induces meristem formation in the germinating seedling or portion thereof; (b) introducing into the cells, via the Agrobacterium, the nucleic acid comprising one or more sequences that, when expressed, modify the cells to achieve the one or more genetic modifications of interest; and (c) culturing the meristem induced by the one or more developmental regulators, to obtain modified plant tissue comprising the one or more genetic modifications of interest. The method can include introducing nucleic acid encoding two or more developmental regulators, wherein the two or more developmental regulators are encoded by one T-DNA or by separate T-DNAs. The method can include introducing nucleic acid encoding two or more developmental regulators, where the two or more developmental regulators are encoded by separate strains of Agrobacterium. The germinating seedling or portion thereof can be from a monocotyledonous plant or from a dicotyledonous plant. The one or more genetic modifications can include insertion of a transgene that, when expressed, achieves an agriculturally relevant trait (e.g., herbicide tolerance). The one or more genetic modifications can include insertion of a transgene that, when expressed, edits the plant cell DNA. The nucleic acid that modifies a plant cell can encode a targeted endonuclease (e.g., a meganuclease, zinc finger nuclease, transcription activator-like effector nuclease, or Clustered Regularly-Interspaced Short Palindromic Repeats-associated nuclease). The nucleic acid that modifies a plant cell can encode a targeted enzyme that modifies plant DNA (e.g., a cytosine deaminase or an adenosine deaminase, such as BE3 or ABE). The nucleic acid that modifies a plant cell can encode a targeted endonuclease and can include a repair template to introduce a specific modification into the genetic material of the plant cell. The method can further include assaying the meristem induced by the one or more developmental regulators for the one or more genetic modifications of interest, and subsequently generating a whole plant from the meristem induced by the one or more developmental regulators. The method also can include placing the meristem induced by the one or more developmental regulators directly into culture and inducing the meristem in culture to form a plant.
In another aspect, this document features a method for generating plant tissue containing one or more genetic modifications of interest. The method can include (a) using Agrobacterium, introducing into cells of a germinating seedling or a portion thereof nucleic acid encoding one or more developmental regulators, wherein expression of the one or more developmental regulators induces meristem formation in the germinating seedling or portion thereof; (b) simultaneously introducing into the cells, via the Agrobacterium, nucleic acid that modifies genetic material within the cells to achieve one or more targeted genetic modifications of interest; and (c) culturing the meristem induced by the one or more developmental regulators, to obtain modified plant tissue containing the one or more genetic modifications of interest. The one or more developmental regulators can include, for example, one or more of Baby Boom, Isopentenyl Transferase, Irrepressible Variants of Monopteros, Shoot Meristemless, and Wuschel. The method can include introducing two or more developmental regulators into cells of the germinating seedling or portion thereof, where the two or more developmental regulators are encoded by one T-DNA, or where the two or more developmental regulators are encoded on separate T-DNAs. The method can include introducing two or more developmental regulators into cells of the germinating seedling or portion thereof, where the two or more developmental regulators are encoded by two or more strains of Agrobacterium.
The germinating seedling or portion thereof can be from a monocotyledonous plant or from a dicotyledonous plant. The one or more targeted genetic modifications can include insertion of a transgene that, when expressed, achieves an agriculturally relevant trait (e.g., herbicide tolerance). The one or more targeted genetic modifications can include insertion of a transgene that, when expressed, edits the plant cell DNA. The nucleic acid that modifies a plant cell can encode a targeted endonuclease, such as a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector (TALE) nuclease, or clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) nuclease. The nucleic acid that modifies a plant cell can encode a targeted enzyme that modifies plant DNA. The targeted enzyme can be a cytosine deaminase or an adenosine deaminase (e.g., BE3 or ABE). The nucleic acid that modifies a plant cell can encode a targeted endonuclease and can contain a repair template to introduce a specific modification into the genetic material of the plant cell. The method can further include assaying meristem induced by the one or more developmental regulators for the one or more genetic modifications of interest, and subsequently generating a whole plant from the meristem induced by the one or more developmental regulators. The method can further include placing the meristem induced by the one or more developmental regulators directly into culture and inducing the meristem in culture to form a plant.
In another aspect, this document features a method for generating plant tissue containing one or more genetic modifications of interest. The method can include (a) introducing into plant cells (i) nucleic acid encoding one or more developmental regulators that, when expressed in the plant cells, induce meristem formation from the plant cells, and (ii) nucleic acid comprising one or more sequences that modify a plant cell to achieve one or more genetic modifications of interest; and (b) deriving de novo tissue from plant cells identified as having the one or more genetic modifications of interest. The one or more developmental regulators can include one or more of Baby Boom, Isopentenyl Transferase, Irrepressible Variants of Monopteros, Shoot Meristemless, and Wuschel. The introducing can be by Agrobacterium, and the nucleic acid encoding one or more developmental regulators and the nucleic acid comprising one or more sequences that modify a plant cell can be included on the same T-DNA. Alternatively, the nucleic acid encoding one or more developmental regulators and the nucleic acid comprising one or more sequences that modify a plant cell can be included on separate T-DNAs. The method can include introducing nucleic acid encoding two or more developmental regulators into the plant cells by Agrobacterium, where the two or more developmental regulators are encoded by a single T-DNA, or where the two or more developmental regulators are encoded by separate T-DNAs. The method can include introducing nucleic acid encoding two or more developmental regulators into the plant cells by Agrobacterium, where the two or more developmental regulators are encoded by two or more strains of Agrobacterium. The introducing can include electroporation, nanoparticle delivery, biolistics, particle bombardment, chemical transfection, or viral infection. The method can include transient delivery of the one or more developmental regulators or stable integration of genes encoding the one or more developmental regulators into the plant genome by any of the above means of delivery.
The plant cells into which the nucleic acids are introduced can be within a differentiated tissue, or within an undifferentiated tissue. The plant cells into which the nucleic acids are introduced can be within a whole plant, or within a plant part taken from a plant. The plant cells can be of a monocotyledonous plant or a dicotyledonous plant.
The one or more sequences that modify a plant cell can include a transgene that, when expressed in the plant cells, achieves an agriculturally relevant trait (e.g., herbicide tolerance). The one or more sequences that modify a plant cell can include a transgene that, when expressed, edits the plant DNA. The one or more sequences that modify a plant cell can include a nucleotide sequence encoding a targeted endonuclease, such as a meganuclease, ZFN, TALE nuclease, or CRISPR/Cas nuclease. The one or more sequences that modify a plant cell can encode a targeted enzyme that modifies plant DNA (e.g., a cytosine deaminase or an adenosine deaminase, such as BE3 or ABE). The one or more sequences that modify a plant cell can encode a targeted endonuclease and can include a repair template to introduce one or more specific modifications into the plant genome. The de novo tissue can be meristematic and capable of deriving new tissue carrying the one or more genetic modifications of interest. The new tissue can include a branch, a flower, or a root.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
A principle goal of GE techniques is the creation of an editing event in the germline of an organism so that the modification can be transmitted to the next generation. For plants, the germline is produced by reproductive tissues derived from the meristem, instead of from isolated gametophyte cells. Plant meristems are the developmental centers of the plant from which all ensuing plant growth is derived. If these stem cells are edited, all tissues subsequently derived from the meristem should contain the GE event(s) of interest, leading to transmission to the next generation. Direct modification of existing meristematic tissue has proven challenging, as it is a highly regulated tissue type that has historically been recalcitrant to genetic modification. This little understood barrier, among other factors, has necessitated the use of suboptimal tissue culture procedures for most agriculturally relevant crops.
The present document is based, at least in part, on the discovery that developmental regulators can be combined with GE reagents to promote the formation of edited meristematic tissue that can flower and produce seed. The methods described herein include steps for delivery of developmental regulators to whole plants to induce the transdifferentiation of somatic plant cells in vivo, leading to the production of meristems. These meristems can carry transgenic insertions or genetic editing events to the next generation, creating seed with a GE event of interest in a fraction of the time needed using current standard protocols. Because the seed is derived from a single meristematic cell, it represents a clonal genetic editing event that provides an abundance of edited seed after a single generation. The direct delivery methods described herein also provide the advantage of avoiding tissue culture, which can reduce the time needed to regenerate tissues and considerably simplifies the process of generating GE events. Thus, the methods provided herein can circumvent the limitations of current plant regeneration protocols, and greatly enhance the potential for development of GE plant lines for both commercial use and basic research.
In some embodiments, this document provides methods in which plants can be grown to a desired stage in either sterile or non-sterile conditions (e.g., soil). In these methods, one or more developmental regulators can be delivered to select tissues, either by Agrobacterium or through ectopic means such as direct injection, electroporation, particle bombardment, biolistics, chemical transfection, viral infection, nanoparticle delivery, or any other suitable means for transient transfection or stable integration (exemplified in
Non-limiting examples of developmental regulators that can be used in the methods provided herein are listed in TABLE 1. As used herein, a “developmental regulator” (DR) is an agent (e.g., a transcription factor, an enzyme, or a hormone) that directs or influences a plant's development, and may guide the differentiation of plant cells, organs, or tissues. For example, a DR can be a transcription factor (e.g., Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, or Wuschel) that can stimulate plant hormone biosynthesis or plant susceptibility to/sensing of cytokinins or other plant hormones that affect plant development and lead to de novo meristem development. In some cases, a DR can lead to increased cytokinin levels. Therefore, a DR also can be a means of increasing one or more cytokinins through ectopic application or through endogenous biogenesis, such as by increasing the expression of one or more enzymes involved in the synthesis of plant hormones. Thus, in some cases, a DR can be an enzyme involved in synthesis of plant hormones, such as Isopentenyl Transferase, which is in the cytokinin biosynthesis pathway. Other examples of enzymes that can lead to increased cytokinin levels and may be useful as DRs include, without limitation, tRNA-isopentenyltransferase, cytochrome P450 monooxygenase, LONELY GUY, adenosine kinase, and adenine phosphoribosyltransferase. A nucleic acid encoding a DR also is considered to be a DR for the purposes of this document, since the nucleic acid can be delivered to plant cells (e.g., in a whole plant or plant part) in order to increase the level of the encoded DR. The DR coding sequence can be operably linked to a promoter (e.g., Nos, 35S, CmYLCV, AtUBQ10, or any other appropriate promoter) that drives expression of the DR in plant cells. Moreover, in some cases, a DR can be a means of increasing expression of genes downstream of the DRs listed in TABLE 1.
Thus, in some embodiments of the methods provided herein, one or more of the DRs listed in TABLE 1 can be delivered to a plant or a plant part.
Exemplary sequences for at least some of the above-referenced DRs and promoters are provided in the attached sequence listing. It is to be noted, however, that homologs of these DRs exist in numerous plant species, and the methods provided herein are not limited to use of the listed DRs or to DRs having 100% identity to the provided sequences. In some cases, for example, a DR coding sequence can have at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identity to the WUS sequence set forth in SEQ ID NO:6, the STM sequence set forth in SEQ ID NO:7, the MPΔ sequence set forth in SEQ ID NO:8, the BBM sequence set forth in SEQ ID NO:9 or SEQ ID NO:10, or the IPT sequence set forth in SEQ ID NO:11. Further, in some cases, a DR can have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to the WUS sequence set forth in SEQ ID NO:118, the STM sequence set forth in SEQ ID NO:119, the MPΔ sequence set forth in SEQ ID NO:120, the BBM sequence set forth in SEQ ID NO:121, or the IPT sequence set forth in SEQ ID NO:122.
The terms “percent identity” or “identity” in the context of two or more nucleic acids or polypeptides refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection.
In general, percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid or polypeptide sequences, dividing the number of matched positions by the total number of aligned nucleotides or amino acids, respectively, and multiplying by 100. A matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned sequences. With regard to DR sequences, the total number of aligned nucleotides or amino acids refers to the minimum number of DR nucleotides or amino acids that are necessary to align the second sequence, and does not include alignment (e.g., forced alignment) with non-DR sequences. The total number of aligned nucleotides or amino acids may correspond to the entire DR sequence or may correspond to fragments of a full-length DR sequence.
Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res, 25:3389-3402, 1997) as incorporated into BLAST® (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST® searches or alignments can be performed to determine percent sequence identity between a DR nucleic acid or amino acid sequence and any other sequence or portion thereof using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST® programs to calculate the percent identity between a NOTCH sequence and another sequence, the default parameters of the respective programs are used.
This document also provides methods that are referred to herein as “Fast-TrACC” (Treated Agrobacterium Co-Culture) methods (exemplified in
Both types of methods described herein can offer new and broadly applicable approaches to solve current bottlenecks in delivery of GE reagents, as well as the regeneration of tissues carrying GE events of interest. Inherently, in vivo delivery of developmental regulators provides a means to easily deliver reagents to somatic tissues, and also provides positive selection for cells receiving GE reagents. By inducing transdifferentiation of somatic cells and subsequent growth on existing tissues, the significant periods of time that otherwise would be necessary for regeneration and development of whole plant tissues can be avoided. Additionally, as these developmental regulators are evolutionarily conserved, these method are amenable to use across a variety of species. Finally, the methods may avoid regulatory hurdles in the development of agricultural crops, as there is potential for transient delivery of reagents and subsequent recovery of non-transgenic progeny carrying a GE event of interest.
Thus, in some embodiments, this document provides methods for generating plant cells, plant parts, plant tissues, or plants that contain one or more genetic modifications of interest, where the methods can include removing existing meristems from a plant, and then introducing nucleic acids into cells of the remaining plant. The introduced nucleic acid sequences can (1) encode one or more (e.g., two, three, four, or more) developmental regulators such as WUS, BBM, IPT, MPA, and/or STM to induce meristem formation, and (2) edit endogenous sequences within the plant cells, or encode polypeptides that act to edit endogenous sequences within the plant cells, to result in a genetic modification of interest. As a result of introducing these nucleic acid sequences, de novo tissue subsequently derived from the plant can carry the genetic modification of interest. In some cases, the de novo tissue can be meristematic, and capable of deriving new tissue (e.g., branch, flower, or root tissue) carrying the genetic modification(s) of interest.
The nucleic acids provided to the plant (e.g., after removal of existing meristems) can be delivered by any suitable method, including by Agrobacterium—in which case the developmental regulator(s) and the editing sequence(s) can be delivered on the same T-DNA or on separate T-DNAs. In some cases, the nucleic acids can be delivered by direct injection, electroporation, biolistics, nanoparticle delivery, particle bombardment, chemical transfection, viral infection, or any other useful method that can result in transient expression or stable integration of the delivered nucleic acid sequences. When two or more developmental regulators are delivered by Agrobacterium, they can be present on the same T-DNA or on separate T-DNAs. In some cases, different strains of Agrobacterium can be used to deliver the developmental regulator(s) and the gene editing component(s). In addition, it is to be noted that the T-DNA(s) used in the methods provided herein can include any suitable replicon. In some cases, for example, a T-DNA can include a viral replicon (e.g., a geminivirus replicon), which can include any appropriate virus component (e.g., RepA) to enable the generation of meristems.
In some embodiments, this document also provides methods for generating plant cells, plant parts, plant tissues, or plants that contain a genetic modifications of interest, where the methods include using Agrobacterium to introduce nucleic acids into germinating seedlings. The nucleic acids can (1) encode one or more (e.g., two, three, four, or more) developmental regulators such as WUS, BBM, IPT, MPΔ, and/or STM to induce meristem formation in the germinating seedling, and (2) edit endogenous sequences within cells of the seedlings, or encode polypeptides that act to edit endogenous sequences within cells of the seedlings, to result in a genetic modification of interest. The methods can further include culturing meristem generated as a result of expression of the developmental regulator(s), to yield modified plant cells, plant tissue, plant parts, and/or plants that contain the genetic modification of interest.
The developmental regulator(s) and the editing nucleic acid(s) can be introduced into the seedling via the same T-DNA or via separate T-DNAs, or even via different strains of Agrobacterium. Similarly, when two or more developmental regulators are used, they can be introduced into the seedling via the same T-DNA, or via separate T-DNAs or different strains of Agrobacterium.
The methods provided herein can be used with monocotyledonous plants, plant cells, plant tissues, and plant parts (e.g., banana, grasses such as Brachypodium distachyon), wheat, oats, barley, maize, Haynaldia villosa, millet, palms, orchids, onions, pineapple, rice, rye, sorghum, and sugarcane) and dicotyledonous plants, plant cells, plant tissues, and plant parts (e.g., alfalfa, amaranth, Arabidopsis, beans, Brassica, carnations, chrysanthemums, citrus plants, coffee, cotton, eucalyptus, grape, impatiens, melons, peanuts, peas, peppers, Petunia, poplars, potatoes, rapeseed, roses, safflower, soybeans, squash, strawberry, sugar beets, sunflower, tobacco, tomatoes, and woody tree species).
In some cases, the methods provided herein can be used to obtain plants, plant tissues, plant parts, and plant cells having a desired trait, such as an agriculturally relevant trait. Agriculturally relevant traits can include, without limitation, herbicide tolerance, resistance to diseases and pests, growth rate, size, shape, color, and flavor of harvested products. For example, the methods provided herein can be used to insert a transgene into the genomic sequence of a plant cell, where expression of the transgene yields the desired trait. In some cases, expression of an inserted transgene can produce a polypeptide that edits the plant DNA. Examples of such polypeptides include targeted rare-cutting endonucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) endonucleases, and RNA-guided endonucleases such as clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) endonucleases), as well as targeted cytosine or adenosine deaminases (e.g., apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-CRISPR/Cas fusions such as BE3, and ABE). Methods for making and using such targeted DNA modifying enzymes are described elsewhere. See, e.g., Sander et al., Nature Methods, 8:67-69, 2011; Jacoby et al., Nucl. Acids Res., 10.1093/nar/gkr1303, 2012); Christian et al., Genetics, 186:757-761, 2010; U.S. Publication No. 2011/0145940; Cong et al., Science 339:819-823, 2013; and Mali et al., Science 339:823-826, 2013. For example, CRISPR/Cas systems use RNA base pairing to direct DNA or RNA cleavage by a Cas endonuclease. CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) sequences direct the Cas enzyme to a specific target DNA sequence (Makarova et al., Nat Rev Microbiol, 9(6):467-477, 2011). The modification of a single targeting RNA can be sufficient to alter the nucleotide target of a Cas protein. In some cases, crRNA and tracrRNA can be engineered as a single cr/tracrRNA hybrid to direct Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012).
In some embodiments, a repair template also can be delivered to plant cells along with a targeted endonuclease. When the endonuclease cleaves the plant cell DNA, the repair template can become integrated into the plant cell's genomic DNA, thus introducing a specific modification into the plant genome.
The methods provided herein also can include culturing the meristem induced by the developmental regulator(s) to give rise to a plant. In some cases, before a plant is generated from the new meristem, de novo derived tissue resulting from expression of the developmental regulator(s) can be assessed to determine whether it includes the genetic modification of interest. For example, DNA from newly derived tissue can be isolated and assessed by restriction digest, hybridization methods (e.g., Southern blotting), or sequencing to determine whether a genetic modification has occurred at the target site. In some embodiments, the expression of a reporter delivered with the developmental regulator(s) and the editing sequence(s) can first be detected, to identify tissues that are likely to carry the genetic modification.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
To facilitate the expression of developmental regulators in plants, T-DNA vectors encoding different arrangements of developmental regulators were generated (SEQ ID NOS:6-11. These T-DNA vectors were designed to have two developmental regulators combined on one T-DNA (SEQ ID NOS:26-32) or to contain a single developmental regulator (SEQ ID NOS:33-37). Construct descriptions are provided in TABLE 2. Developmental regulators were expressed using the 35S (SEQ ID NO:1), CmYLCV (SEQ ID NO:3), AtUBQ10 (SEQ ID NO:2), or Nos (SEQ ID NO:5) promoter. For Fast-TrACC experiments, these vectors often coexpressed the RNA guided endonuclease Cas9 (SEQ ID NO:24) driven by the 35S promoter. For many of the experiments, a gRNA was expressed under the control of an AtU6 promoter (SEQ ID NO:4) targeting both of the duplicated PDS1 homologs (Niben101Scf14708g00023.1, SEQ ID NO:16; and Niben101Scf01283g02002.1, SEQ ID NO:17) in the N. benthamiana genome. A luciferase reporter gene (SEQ ID NO:14) driven by either the 35S or the CmYLCV promoter was used as a visual confirmation of construct delivery. These constructs were cloned into the T-DNA backbone of pTRANS_201 (SEQ ID NO:18) or pTRANS_221 (SEQ ID NO:19) as described elsewhere (Cermak et al., Plant Cell 29(6):1196-1217, 2017). This Agrobacterium vector was designed to deliver a T-DNA containing a modified bean yellow dwarf virus (BeYDV) capable of circularization and replication upon delivery and expression of the encoded replication protein (Rep; SEQ ID NO:25) in vivo. Replication of the replicon can enable an increased copy number of the vector, and consequently high levels of gene expression. Additionally, this vector has the potential to replicate regardless of whether it integrates into the genome, enabling transient high copy delivery of vector constructs.
Plant cells are inherently totipotent and can be transdifferentiated into other cell types. Thus, studies were conducted to determine whether de novo meristems could be induced in vivo by ectopically expressing DRs in plant somatic cells, and by co-delivering gene editing reagents with the DRs, whether it might be possible to create edited meristems that ultimately produce seed and transmit induced genetic changes to the next generation.
Young (10-12 week-old) transgenic N. benthamiana plants that constitutively expressed Cas9 were pruned to remove all visibly discernible shoot meristems (
To demonstrate that transgenic tissues were generated de novo from injected tissues, samples were visualized for luciferase activity. Tissues were isolated from newly formed shoots arising from Agrobacterium injection sites. To visualize luciferase expression, tissues were immersed in water containing 5 mM luciferin for 5 minutes prior to bright field imaging or imaging in the dark using a CCD camera. Some tissues demonstrated luciferase expression, indicating that the newly formed tissues were derived from cells that had received the T-DNA from Agrobacterium (
In subsequent studies, transgenic shoots are allowed to develop and produce flowers. At some frequency, transgenes are transmitted to progeny, thereby creating stably transgenic plants.
The T-DNA delivered to the transgenic Cas9 plants also expressed a gRNA targeting N. benthamiana phytoene desaturase (PDS) genes. There are two PDS homologs in N. benthamiana (NbPDS1; Niben101Scf14708g00023.1 and NbPDS2; Niben101Scf01283g02002.1). The gRNA was selected to target conserved sequences in both genes. In the absence of PDS, photobleaching occurs due to lack of photoprotective carotenoids, giving rise to a readily discernible phenotype (Qin et al., Cell Res 17:471-482, 2007). A subset of the shoots that emerged in the experiments described in Example 2 were white, suggesting biallelic inactivation of the two PDS homologs (
To confirm the creation of non-chimeric, genetically modified de novo tissue, genomic DNA was isolated from tissues exhibiting the PDS phenotype. NGS primers specific to the Scf14708g00023.1 homolog (NbPDS1, SEQ ID NOS:78-87) were used to amplify the locus in the genomic DNA sample, and the resulting amplicon was submitted for Illumina sequencing. The results demonstrated a mutation profile consistent with a single editing event at the target locus as compared to negative controls (
It was desired to determine whether GE in induced shoots could transmit the edits to the next generation. However, none of the shoots with developmental abnormalities or the PDS phenotype set seed. Because all shoots were molecularly surveyed for mutations at the PDS targets, however, one green shoot that produced viable seed and had a 3 bp deletion in one PDS allele was identified (
One shoot emerged that was chimeric for white and green tissue, but otherwise was phenotypically normal and non-bioluminescent (
To determine if de novo meristems could be induced on agronomically important species, additional experiments were performed in Vitis vinifera (grape) and Solanum tuberosum (potato). Cuttings from asexually propagated potato (
In further studies, transgenic shoots are created in potato, grape, or other species that express Cas9 and a gRNA targeting an endogenous gene. Cas9 and the gRNA create mutations in somatic cells, which are induced by the DRs to form meristems and shoots. Some shoots have edited genes that produce flowers and transmit gene edits to the next generation. Other shoots have edited genes but lack the transgene and produce edited, transgene-free progeny (as described in Example 4 for N. benthamiana, for example).
Examples 1-5 describe studies using methods to generate de novo meristems in whole plants that either carry transgenes or have GE events. This Example and the following Examples describe studies showing that de novo meristem-like tissue can be generated out of somatic tissue via Fast-TrACC delivery of developmental regulators (
Fast-TrACC involves treating A. tumefaciens cultures (GV3101 was used in the studies described herein, but any other suitable strain can be used) for three days prior to a two day co-culture with newly germinated seedlings. The first step is to grow the cultures overnight (8-12 hours) at 28° C. Next, cells are harvested by centrifugation and re-suspended to an OD600 of 0.3 in AB:MES salts (17.2 mM K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM KCl, 1.25 mM MgSO4, 100 μM CaCl2, 10 μM FeSO4, 50 mM MES, 2% glucose (w/v), 200 μM acetosyringone, pH 5.5) (Wu et al., Plant Methods 10:19, 2014), with the intent to increase the expression of vir genes, and then grown overnight. The culture is again centrifuged and resuspended to OD600 within the range of 0.10 to 0.18 in a 50:50 (v/v) mix of AB:MES salts and ½ MS liquid plant growth medium (½ MS salt supplemented with 0.5% sucrose (w/v), pH 5.5). The A. tumefaciens culture is now ready for incubating with seedlings.
Seeds are sterilized using 70% ethanol for 1 minute and 50% bleach (v/v) for 5 minutes. The seeds are then rinsed 5 times with sterile water, and transferred to 6-well plates (˜5 seeds per well in 2 mL ½ MS), where they are subsequently germinated and maintained in growth chambers for 2-3 days at 24° C. under a 16 hour/8 hour light/dark cycle. A. tumefaciens is added and the co-culture is incubated for two days before the seedlings are washed free of A. tumefaciens using sterile water. The washed seedlings are returned to liquid ½ MS containing 100 μM of antibiotic timentin to effectively counter-select against residual A. tumefaciens.
The Fast-TrACC method was used to deliver Agrobacterium tumefaciens T-DNA constructs (SEQ ID NOS:26 and 27) encoding developmental regulators into the cotyledons of N. benthamiana seedlings. These constructs contained a luciferase reporter (CmYLCV:Luc, SEQ ID NO:3:SEQ ID NO:14) and the developmental regulators WUS (Nos:WUS, SEQ ID NO:5:SEQ ID NO:6) and STM (SEQ ID NO:7). STM was expressed using three different promoters (35S, SEQ ID NO:1; AtUBQ10, SEQ ID NO:2; and CmYLCV, SEQ ID NO:3) to determine if one had optimal meristem patterning potential. All three promoters formed growths but at slightly different frequencies.
Using the luciferase reporter expression as a proxy for T-DNA delivery, sectors of the plant that received the developmental regulators were identified. From regions exhibiting high levels of localized luciferase expression (
To determine the ability to produce plants from de novo growths, N. benthamiana seedlings with meristem-like growths were selected as candidates for the generation of transgenic plants. The meristem-like growths were derived using the developmental regulator combination WUS and STM. Efforts were made to ensure that the newly formed meristem-like tissue could be propagated. The meristem-like tissues were grown for about 3 weeks, until they started to form secondary leaves (
One purpose of generating transgenic plants, whether of mosaic or uniform genetic constitution, was to produce flowers. These flowers can be derived from the same tissues that lead to the formation of luciferase positive leaves, implying that at some frequency, the flowers have the T-DNA and therefore will be transgenic themselves. The transgenic flowers will produce seed, some of which will be transgenic. Seeds from transgene-positive plants can be collected and tested for luciferase expression (i.e., presence of the transgene) and for the presence of GE events. Demonstration of heritable transmission of transgenes is described below in Example 10; demonstration of heritable transmission of GE events is described below in Example 12.
To demonstrate that GE events can occur in de novo growths, gene editing reagents were combined with developmental regulators and delivered to seedlings. The construct that was used (SEQ ID NO:29) contained 35S:Cas9 (SEQ ID NO:1:SEQ ID NO:24), AtU6:gRNA (SEQ ID NO:4:SEQ ID NO:12), Nos:WUS (SEQ ID NO:5:SEQ ID NO:6), and CmYLCV:STM (SEQ ID NO:3:SEQ ID NO:7). Limits on construct size prevented the inclusion of luciferase as a delivery reporter. Seedlings were screened for production of growths. Out of twenty-four seedlings, five seedlings exhibited growth formations (
The growths were excised and DNA was isolated from each. From the isolated DNA, the target region in NbPDS1 was PCR amplified. The gRNA used in these studies targeted a locus with a NcoI restriction site that, when edited, will not allow for digestion in a RFLP assay. Four of the five tested samples contained the “protected” band indicative of editing (
To illustrate the capability to regenerate edited plants in a fashion similar to the generation of transgenic plants, whole plants were derived from edited growths. The same construct (SEQ ID NO:29) used to generate edits within undifferentiated growths was used to promote the formation of plantlets with edits. A new set of growths were developed on N. benthamiana cotyledons that then formed meristem-like growths. Once the growths established a shoot-like structure, they were transferred to rooting medium to initiate a root network. Full plants were considered formed once the root system was established (
Leaf tissue samples were taken from the generated plants and submitted for NGS. The sequencing results from individual tissue samples resulted in a small proportion of edited reads (
Ideally, these chimeric plants will contain mutations within a floral meristem, which would cause the flower to produce edited seeds at some frequency. As described below in Example 12, plants are grown and seeds are collected and tested for gene editing. The isolation of edited plants derived from these seeds demonstrates that edited plants can be obtained in a single generation through the creation of developmental regulator-derived plants.
The ultimate goal for DR-based plant generation is to create genetic changes that can be transmitted to subsequent generations. Several of the DR-derived N. benthamiana plants grew seed-bearing flowers. These plants were created using the constructs pRN114, pRN119 and pRN120 (SEQ ID NOS:26-28) and exhibited different levels of luciferase expression (
Fast-TrACC was used to test different combinations of developmental regulators in order to identify those that could best induce growths that give rise to full plants. Separate A. tumefaciens strains, each carrying expression cassettes for a unique DR, were pooled for seedling co-culture. Twelve combinations of DRs were tested, and five of those combinations resulted in growths from which plants could be derived (
Studies were conducted to determine if Fast-TrACC could be used to generate meristems with gene edits and subsequently plants that transmit mutations to progeny. In the experiment described in Example 11, transgenic N. benthamiana seedlings constitutively expressing Cas9 were treated with Fast-TrACC. In addition to a DR, the T-DNAs carried a cassette that expressed a gRNA targeting NbPDS1 and NbPDS2. Biallelic knockouts of both PDS homologs are expected to result in a white phenotype due to chlorophyll photobleaching (Qin et al., supra). About 15% of the generated shoots showed evidence of photobleaching, but these shoots did not form full plants; they were likely compromised by lack of chlorophyll (
Of 27 total plants recovered in the experiment described in Example 11, five phenotypically normal green plants were found to show considerable amounts of editing in somatic cells (
As DRs are evolutionarily conserved, studies were conducted to determine whether the approach for seedling transformation would be applicable to other plant species. Combinations of DRs that generated de novo meristems on N. benthamiana seedlings were therefore tested to determine whether they could induce shoots on other dicots, such as tomato. In particular, Fast-TrACC was used to deliver three combinations of developmental regulators (Nos:WUS&35S:STM, Nos:WUS&CmYLCV:STM, and Nos:WUS&35S:IPT) to tomato seedlings. For both combinations of WUS & STM, no shoot-like growths formed (
Next, WUS and IPT were delivered to tomato seedlings on either a single vector (WUS/IPT) or on separate vectors in two different Agrobacterium strains (WUS&IPT). Both WUS/IPT and WUS&IPT showed an increase in the frequency of average growths per plant over the background level of callus-like growths that developed on plants that did not receive developmental regulators (
In subsequent experiments, transgenic shoots are placed on root-inducing medium to promote root formation. Resulting plantlets are transferred to soil where they continue to grow, flower, and produce fruit and seed. Progeny are assessed to for transmission of the transgene, as demonstrated in Example 10. Gene edited tomato plants are generated and assessed through an approach similar to that described in detail in Example 12 for N. benthamiana.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a National Stage application under 35 U.S.C. § 371 of International Application No. PCT/US2019/039297 having an International Filing Date of Jun. 26, 2019, which claims priority from U.S. Provisional Application Ser. No. 62/690,165, filed Jun. 26, 2018. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
This invention was made with government support under IOS-1339209 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/039297 | 6/26/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/006112 | 1/2/2020 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7473822 | Paz et al. | Jan 2009 | B1 |
| 8586363 | Voytas et al. | Nov 2013 | B2 |
| 20110145940 | Voytas et al. | Jun 2011 | A1 |
| 20140157453 | Gordon-Kamm et al. | Jun 2014 | A1 |
| 20150059010 | Cigan et al. | Feb 2015 | A1 |
| 20150166980 | Liu et al. | Jun 2015 | A1 |
| 20150166981 | Liu et al. | Jun 2015 | A1 |
| 20150167000 | Voytas et al. | Jun 2015 | A1 |
| 20160237451 | Voytas et al. | Aug 2016 | A1 |
| 20170121722 | Anand et al. | May 2017 | A1 |
| 20180051267 | Voytas et al. | Feb 2018 | A1 |
| 20190177740 | Gou et al. | Jun 2019 | A1 |
| 20190249183 | Humanes et al. | Aug 2019 | A1 |
| 20210047652 | Zhang et al. | Feb 2021 | A1 |
| 20210054389 | Cho et al. | Feb 2021 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2002004649 | Jan 2002 | WO |
| WO 2017112006 | Jun 2017 | WO |
| WO 2017123772 | Jul 2017 | WO |
| WO 2018080389 | May 2018 | WO |
| Entry |
|---|
| Gallois et al., 2002, Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis, Development 129: 3207-3217. |
| Mishra and Zhao, 2018, Genome editing technologies and their applications in crop improvement, Plant Biotechnology Reports, 12: 57-68. |
| Rodríguez-Leal et al., 2017, Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing, Cell 171: 470-480. |
| Li et al., 2016, Generation of Targeted Point Mutations in Rice bya Modified CRISPR/Cas9 System, Molecular Plant 10: 526-529. |
| Liu et al., 2018, Phenotypic novelty by CRISPR in plants, Developmental Biology 435: 170-175. |
| Gallois, J.L. et al, Development (2002) vol. 129, pp. 3207-3217. (Year: 2002). |
| Altpeter et al., “Advancing Crop Transformation in the Era of Genome Editing,” Plant Cell, 28(7):1510-1520, Jul. 2016. |
| Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucl. Acids Research, 25(17):3389-3402, Sep. 1997. |
| Bairu et al., “Somaclonal variation in plants: causes and detection methods,” Plant Growth Regulation, 63(2):147-173, Mar. 2011. |
| Banakar et al., “High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice,” Sci. Reports, 9:19902, Dec. 2019, 13 pages. |
| Barton, “Twenty years on: The inner workings of the shoot apical meristem, a developmental dynamo,” Dev. Biology, 341(1):95-113, May 2010. |
| Butler et al., “Crop improvement using genome editing,” Plant Breeding Reviews, 41:55-101, Jan. 26, 2018. |
| Campbell et al., “Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes,” Sci. Reports, 9:14757, Oct. 14, 2019, 11 pages. |
| {hacek over (C)}ermák et al., “A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants,” Plant Cell, 29(6):1196-1217, Jun. 2017. |
| Christian et al., “Targeting DNA Double-Strand Breaks with TAL Effector Nucleases,” Genetics, 186(2):757-761, Oct. 2010. |
| Ckurshumova et al., “Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation,” New Phytologist, 204(3):556-566, Nov. 2014. |
| Cong et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science, 339(6121):819-823, Feb. 15, 2013. |
| Curtin et al., “CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula,” Plant Biotechnol. Journal, 16(6):1125-1137, Jun. 2018. |
| Curtin et al., “Genome Engineering of Crops with Designer Nucleases,” Plant Genome, 5(2):42-50, Jul. 2012. |
| Curtin et al., “MicroRNA Maturation and MicroRNA Target Gene Expression Regulation Are Severely Disrupted in Soybean dicer-like1 Double Mutants,” G3 (Bethesda), 6(2):423-433, Feb. 2016. |
| Curtin et al., “Targeted Mutagenesis for Functional Analysis of Gene Duplication in Legumes,” Methods Mol. Biology, 1069:25-42, Aug. 2013. |
| Curtin et al., “Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases,” Plant Physiology, 156(2):466-473, Jun. 2011. |
| Curtin et al., “Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation,” Plant Physiology, 173(2):921-931, Feb. 2017. |
| Demorest et al., “Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil,” BMC Plant Biology, 6:225, Oct. 13, 2016, 8 pages. |
| Gallois et al., “Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis,” Development, 129(13):3207-3217, Jul. 2002. |
| Gasiunas et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc. Natl. Acad. Sci. USA, 109(39):E2579-E2586, Sep. 4, 2012. |
| Gelvin, “Agrobacterium-Mediated Plant Transformation: the Biology behind the ‘Gene-Jockeying’ Tool,” Microbiol. Mol. Biol. Reviews, 67(1):16-37, Mar. 2003. |
| Graham et al., “Plant Genome Editing and the Relevance of Off-Target Changes,” Plant Physiology, 183(4):1453-1471, Aug. 2020. |
| Groß-Hardt et al., “Stem cell regulation in the shoot meristem,” J. Cell Science, 116(9):1659-1666, May 2003. |
| Haun et al. , “Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family,” Plant Biotechnology Journal 12(7):934-940, Sep. 2014. |
| Heidstra et al., “Plant and animal stem cells: similar yet different,” Nat. Rev. Mol. Cell Biology, 15(5):301-312, May 2014. |
| Jacoby et al., “Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space,” Nucl. Acids Research, 40(11):4954-4964, Feb. 14, 2012. |
| Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science, 337(6096):816-821, Aug. 17, 2012. |
| Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, 533(7603):420-424, May 19, 2016. |
| Kumaran et al., “YABBY Polarity Genes Mediate the Repression of KNOX Homeobox Genes in Arabidopsis,” Plant Cell, 14(11):2761-2770, Nov. 2002. |
| Kunkel et al., “Inducible isopentenyl transferase as a high efficiency marker for plant transformation,” Nat. Biotechnology, 17(9):916-919, Sep. 1999. |
| Lee et al., “An Overview of Genetic Transformation of Soybean, A Comprehensive Survey of International Soybean Research,” Genetics, Physiology, Agronomy and Nitrogen Relationships, Chapter 23, Jan. 2, 2013, 18 pages. |
| Liang et al., “Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins,” Nat. Protocols, 13(3):413-430, Mar. 2018. |
| Liu et al., “Genome Editing in Soybean with CRISPR/Cas9,” Methods Mol. Biology, 1917:217-234, Jan. 2019. |
| Lowe et al., “Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation,” Plant Cell, 28(9):1998-2015, Sep. 2016. |
| Makarova et al., “Evolution and classification of the CRISPR-Cas systems,” Nat. Rev. Microbiology, 9(6):467-477, Jun. 2011. |
| Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnology, 31:833-838, Aug. 2013. |
| Mali et al., “RNA-Guided Human Genome Engineering via Cas9,” Science, 339(6121):823-826, Feb. 15, 2013. |
| Michno et al., “CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme,” GM Crops Food, 6(4):243-252, Mar. 1, 2016. |
| Mookkan et al., “Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2,” Plant Cell Reports, 36(9):1477-1491, Sep. 2017. |
| PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2019/039297, dated Dec. 29, 2020, 7 pages. |
| PCT International Search Report and Written Opinion in International Appln. No. PCT/US2019/039297, dated Oct. 9, 2019, 10 pages. |
| Qin et al., “Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis,” Cell Research, 17(5):471-482, May 2007. |
| Sander et al., “Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA),” Nat. Methods, 8(1):67-69, Jan. 2011. |
| Schmidt et al., “Towards normalization of soybean somatic embryo maturation,” Plant Cell Reports, 24(7):383-391, Sep. 2005. |
| Somers et al., “Recent Advances in Legume Transformation,” Plant Physiology, 131(3):892-899, Mar. 2003. |
| Southern et al., “Luciferases as Reporter Genes,” Methods Mol. Biol. Arabidopsis Protocols, 323:293-305, 2006. |
| Stupar et al., “All in the Family: Understanding soybean gene redundancies through genome engineering,” Presented at Proceedings of Crops 2015 Conference, Huntsville, AL, May 18-21, 2015, 29 pages. |
| Stupar et al., “Building a better mutant: Challenges and opportunities for understanding and utilizing gene functions in soybean,” Presented at Proceedings of the Université Laval Institute for Integrative and Systems Biology Seminar Series, Quebec City, Canada, Oct. 4-8, 2017, 41 pages. |
| Stupar et al., “Comparison of genomic structural variation associated with cultivars, mutagenized, and transgenic soybeans,” Presented at Proceedings of the Plant and Animal Genome Conference, San Diego, CA, Jan. 10-14, 2015, 22 pages. |
| Stupar et al., “Identification of Functional Variants in Soybean Using Fast Neutron and CRISPR-Based Mutagenesis,” Presented at Proceedings of the Plant and Animal Genome Conference, San Diego, CA, Jan. 13-18, 2017, 27 pages. |
| Stupar, “All in the Family: Understanding soybean gene redundancies through genome engineering,” Presented at Proceedings of the Iowa State University Genome Editing: Foundations and Applications Meeting, Ames, IA, Apr. 9-11, 2015, 39 pages. |
| Stupar, “Applications of engineered nucleases,” Presented at Proceedings of the Soybean Precision Genomics Workshop 2013, Columbia, MO, Jul. 7-10, 2013, 4 pages. |
| Stupar, “Cloudy with a chance of mutations: Gene editing and functional analyses in soybean,” Presented at Proceedings of the 17th Biennial Molecular and Cellular Biology of the Soybean Conference, Athens, GA, Aug. 26-29, 2018, 20 pages. |
| Stupar, “Cloudy with a chance of mutations: Gene editing and functional analyses in soybean,” Presented at Proceedings of the Plant and Animal Genome Conference, San Diego, CA, Jan. 12-16, 2019, 5 pages. |
| Stupar, “CRISPR/Cas-9 overview, applications and case studies,” Presented at Proceedings of the Soybean Precision Genomics and Mutant Finder Workshops 2016, Columbia, MO, Aug. 3-4, 2016, 28 pages. |
| Stupar, “Gene Editing for Crop Improvement,” Presented at Proceedings of the ASA-CSSA-SSSA 2015 International Annual Meetings Symposium, Minneapolis, MN, Nov. 15-18, 2015, 23 pages. |
| Stupar, “Inheritance patterns of transgenes and targeted mutations in a soybean CRISPR-based system,” Presented at Proceedings of the 16th Biennial Molecular and Cellular Biology of the Soybean Conference, Columbus, OH, Aug. 7-10, 2016, 19 pages. |
| Stupar, “Old mutations and new biotechnology: expanding and understanding the genetic resources of soybean,” Presented at Proceedings of the University of York Genomics Seminar, York, UK, Mar. 22, 2016, 63 pages. |
| Stupar, “Opportunities and obstacles for candidate gene validation using CRISPR/Cas in soybean,” Presented at Proceedings of the ASA-CSSA-SSSA 2016 International Annual Meetings Svmposium, Phoenix, AZ, Nov. 6-9, 2016, 21 pages. |
| Stupar, “Opportunities and Obstacles for CRISPR in Soybean: Lessons Learned from the Inheritance of Transgenes and Targeted Mutations,” Presented at Proceedings of the Banbury Center Genomics-enabled Accelerated Crop Breeding Meeting, Long Island, NY, Oct. 16-19, 2016, 31 pages. |
| Stupar, “Opportunities and Obstacles for CRISPR in Soybean: Lessons Learned from the Inheritance of Transgenes and Targeted Mutations,” Presented at Proceedings of the Donald Danforth Plant Sciences Center 18th Annual Fall Symposium, St. Louis, MO, Sep. 28-30, 2016, 30 pages. |
| Stupar, “Opportunities and Obstacles for CRISPR in Soybean: Lessons Learned from the Inheritance of Transgenes and Targeted Mutations,” Presented at Proceedings of the National Association of Plant Breeders Annual Meeting, Raleigh, NC, Aug. 15-18, 2016, 6 pages. |
| Stupar, “Overcoming the bottleneck: Understanding and expanding soybean genetic diversity,” Presented at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China, Feb. 16-25, 2014, 42 pages. |
| Stupar, “Soybean breeding with genome-editing technology,” Presented at Proceedings of the 2019 Soybean Breeders Workshop, St. Louis, MO, Feb. 11-13, 2019, 6 pages. |
| Stupar, “Soybean precision genomics,” Presented at Proceedings of the Soybean Precision Genomics Workshop 2012, St. Paul, MN, Jul. 18-20, 2012, 23 pages. |
| Stupar, “The CRISPR x Genome interaction: Challenges and opportunities for understanding gene function in soybean,” Presented at Proceedings of Biotechnology Havana 2017, Havana, Cuba, Dec. 3-6, 2017, 29 pages. |
| Stupar, “USDA-BRAG: Genomic variation associated with different soybean germplasm sources,” Presented to the U.S. Department of Agriculture—Animal and Plant Health Inspection Service, Washington, DC, Sep. 2016, 23 pages. |
| UniProt Accession No. Q03JI6, “CRISPR-associated endonuclease Cas9 2,” dated May 23, 2018, 3 pages. |
| UniProt Accession No. Q99ZW2, “CRISPR-associated endonuclease Cas9/Csn1,” dated Jun. 20, 2018, 11 pages. |
| Veena et al., “Agrobacterium rhizogenes: recent developments and promising applications,” In Vitro Cell. Dev. Biol.—Plant, 43(5):383-403, Nov. 2007. |
| Virdi et al., “Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog,” Front. Plant Science, 11:1005, Jul. 2020, 11 pages. |
| Voytas, “Genome Editing and Plant Agriculture: Expression of Developmental Regulators for Accelerated Plant Gene Editing,” Presentation at the Federation of American Societies for Experimental Biology Science Research Conference—Genome Engineering: Cutting Edge Research and Applications, Florence, Italy, Jun. 24-28, 2017, 26 pages. |
| Wu et al., “AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings,” Plant Methods, 10:19, Jun. 2014, 16 pages. |
| Zeng et al., “Refined glufosinate selection in Agrobacterium mediated transformation of soybean [Glycine max (L.) Merrill],” Plant Cell Reports, 22(7):478-482, Feb. 2004. |
| U.S. Appl. No. 16/913,478, filed Jun. 26, 2020, Feng Zhang, Published as U.S. 2021/0047652. |
| Boehm (2014) (Masters Theses, Univ. Tenn.) “Molecular Marker Assisted Backcross Development and Evaluation of an Environmentally Friendly, Commercially Acceptable Low Seed Phytate Soybean.” |
| Number | Date | Country | |
|---|---|---|---|
| 20210269813 A1 | Sep 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62690165 | Jun 2018 | US |
