Improving pennycress traits to increase productivity

Abstract

Genetically modified pennycress plants that exhibit improved response to shade and higher temperatures are disclosed. Nucleic acid constructs and methods of using the nucleic acid constructs for producing the genetically modified pennycress plants are also disclosed.

Description

IMPROVING PENNYCRESS TRAITS TO INCREASE PRODUCTIVITYGOVERNMENTAL RIGHTS

[0001] This invention was made with government support under DE-SC0021286 awarded by the U.S. Department of Energy. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from Provisional Application number 63 / 550,436, filed February 6, 2024, the entire contents of which are hereby incorporated by reference.INCORPORATION OF SEQUENCE LISTING

[0003] The present application contains a Sequence Listing that has been submitted in .XML format via Patent Center and is hereby incorporated herein by reference in its entirety. Said .XMI was created on February 3, 2025, is named Sequence_listing_DDPSC0146, and is 33 kilobytes in size.FIELD OF THE INVENTION

[0004] The present disclosure provides genetically modified pennycress plants exhibiting improved response to shade and higher temperatures, compositions, and methods of generating the plants.BACKGROUND OF THE INVENTION

[0005] Much of the 80 million-acre U.S. Midwest Corn Belt lay fallow each winter. Planting an off-season crop could extend the growing season, protect soils, and reduce water pollution by scavenging excess nutrients. Pennycress (Thlaspi arvense, field pennycress) is under development as a new winter annual bioenergy oilseed to be grown as a third crop in the Midwest. Pennycress oil has a fatty acid composition well-suited for conversion to biodiesel and bio jet fuel that meets the U.S. Renewable Fuels Standard. Academic, governmental, and industrial stakeholders are working closely to rapidly commercialize domesticated pennycress varieties that can yield over 1680 kg ha'1(1500 lb ac1) of seeds producing 600 liters ha-1 (65 gal ac1) of oil annually without competing with food crops. A specific scenario involves inter-seeding pennycress with corn, where pennycress is planted in late fall into standing corn fields. This practice can maximize land use efficiency by utilizing the fallow period between corn harvest and the next planting season. However, crucial work remains to domesticate and optimize pennycress for incorporation into present cropping systems and its resilience to climate change. For example, inter-seeding into standing fields in late fall can lead to shade- induced elongation responses in pennycress. Similarly, higher temperatures during planting can also cause seedlings to elongate. Excessive elongation creates a cyclical dilemma, in which the elongated plants increasingly shade their neighbors, further inducing retaliatory elongation responses in efforts to outgrow neighboring plants.These morphogenic changes are undesirable in cropping systems as elongated plants establish poorly, are more prone to lodging, and typically reduce yield.

[0006] Accordingly, there is a need for pennycress plants adapted for incorporation into present cropping systems as an off-season crop, including improved responses to shade and higher temperatures.SUMMARY OF THE INVENTION

[0007] One aspect of the instant disclosure encompasses a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response to shade and higher temperatures. The genetically modified plant comprises a genetic modification of at least one target nucleic acid sequence, wherein the genetic modification modifies expression of a phytochrome-interacting factor 7 (PIF7) protein, thereby conferring an improved response to shade and higher temperatures to the Thlaspi sp. plant.

[0008] In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95%or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The genetic modification can be in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetic modification is in an endogenous nucleic acid sequence encoding the PIF7 protein. The genetic modification can be a loss of function mutation.

[0009] In some aspects, the Thlaspi sp. plant is Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense. In some aspects, the Thlaspi sp. plant is Thlaspi arvense. The genetic modification that modifies expression of the PIF7 protein can be a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein. The endogenous nucleic acid sequence comprising the loss of function mutation can comprise at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or any combination thereof.

[0010] In some aspects, the modification of the polynucleotide encoding the PIF7 protein comprises a modification introduced by a programmable nucleic acid modification system comprising a CRISPR / Cas nuclease system, wherein the CRISPR / Cas nuclease system comprises a CAS9 protein (CAS9) and a guide RNA (gRNA). In some aspects, the Thlaspi sp. is Thlaspi arvense and the gRNA comprises a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof.

[0011] Another aspect of the instant disclosure encompasses one or more expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed. The one or more expression constructs comprise a promoter operably linked to a nucleic acid sequence encoding a programmable nucleic acid modification system targeted to a target nucleic acid sequence. Expression of the nucleic acid modification system in the plant or part thereof, plant cell, or seed introduces a genetic modification in the target nucleic acid sequence, thereby conferring an improved response to shade and higher temperatures to the Thlaspi sp. plant.

[0012] In some aspects, the Thlaspi sp. plant is Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense. In some aspects, the pennycress plant is Thlaspi arvense (pennycress). The programmable nucleic acid modification system can comprise a CRISPR / Cas nuclease system, wherein the CRISPR / Cas nuclease system comprises a CAS9 protein (CAS9) and a guide RNA (gRNA). The Cas9 nuclease can be encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with an amino acid sequence of SEQ ID NO: 8.

[0013] In some aspects, the Thlaspi sp. is Thlaspi arvense and the gRNA comprises a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof. The one or more expression constructs for expressing the CRISPR / Cas9 system can comprise an expression construct for expressing Cas9 nuclease, and an expression construct for expressing the guide RNA. The expression construct for expressing the Cas9 nuclease can comprise a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 9. The expression construct for expressing the gRNA can comprise a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 10.

[0014] Yet another aspect of the instant disclosure encompasses a Thlaspi sp. plant or plant cells comprising one or more expression constructs described herein above.

[0015] An additional aspect of the instant disclosure encompasses a method of generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures. The method comprises introducing one or more expression constructs described herein above into a Thlaspi sp. plant or part thereof, plant cell, or seed; and growing the Thlaspi sp. plant or part thereof, plant cell, or seed for a time and under conditions sufficient for the one or more nucleic acid expression constructs to express the programmable nucleic acid modification system in the Thlaspi sp. plant or part thereof, plant cell, or seed.Expressing the programmable nucleic acid modification system introduces a nucleicacid modification in the nucleic acid sequence encoding a PIF7 protein, thereby modifying the expression of the PIF7 protein, thereby generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising improved response to shade and higher temperatures to the Thlaspi sp. plant.

[0016] One aspect of the instant disclosure encompasses a method of improving response of a Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures. The method comprises generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response to shade and higher temperatures described herein above using a method described herein above.

[0017] Another aspect of the instant disclosure encompasses a method of improving performance of a Thlaspi sp. plant in culture. The method comprises cultivating a genetically modified Thlaspi sp. plant, or a part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures described herein above, thereby improving performance of the Thlaspi sp. plant in culture.Improving performance of a Thlaspi sp. plant comprises improving the yield of a Thlaspi sp. plant in an agricultural system comprising sequential or overlapping cropping cycles, enhancing weed suppression, increased biomass, or any of the combinations thereof.

[0018] Yet another aspect of the instant disclosure encompasses a kit for generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures. The kit comprises one or more genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures described herein above; one or more expression constructs described herein above for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; Thlaspi sp. plant or plant cells described herein above comprising expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0019] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] FIG. 1A is a photograph of pennycress 5-d-old seedlings grown under white light or shade conditions.

[0021] FIG. 1B is a bar plot showing the effect of white light conditions, shade, at 28°C, and a combination of shade and 28°C on hypocotyl length of 5-day-old pennycress seedlings.

[0022] FIG. 1C are photographs of pennycress rosettes of 10-d-old plants exposed to white light or shade for 1 d. n = 6 p < 0.01

[0023] FIG. 1D is a bar plot of rosette solidity of 10-d-old wild-type plants exposed to white light or shade for 1 d. n = 6 p < 0.001 .

[0024] FIG. 1E is a photograph showing bolting in plants grown under shade conditions when compared to plants grown under white light conditions.

[0025] FIG. 1F is a graph showing the percentage of plants with bolt > 1 inch. White light or shade treatment began at day 11. n = 18

[0026] FIG. 1G is a photograph showing the increased height of plants grown under shade conditions when compared to white light conditions.

[0027] FIG. 2 is an illustration showing the loss of function mutations generated in PIF7. Black boxes are exons; introns are a line.

[0028] FIG. 3A is a photograph showing the effect of shade on hypocotyl length in pif7 loss of function plants.

[0029] FIG. 3B is a plot showing the effect of shade on hypocotyl length in pif7 loss of function plants.

[0030] FIG. 4A is a photograph showing rosettes of wt and pif7 mutant plants.

[0031] FIG. 4B are bar plots showing measured solidity, convex hull area, and perimeter of wild type and mutant plants. Convex hull area and perimeter were measured as shown in the photograph.

[0032] FIG. 4C is a bar plot of the convex hull area of rosettes of plants grown under control conditions for 7 d then for 14 d in treatment conditions. Solidity was determined using the shape analysis function from PlantCV. Common letters indicated no significant difference.

[0033] FIG. 4D is a bar plot showing rosette solidity of plants grown under control conditions for 7 d then for 14 d in treatment conditions. Hull area was calculated using the shape analysis function from PlantCV. Common letters indicated no significant difference.

[0034] FIG. 4E are bar plots showing the difference in plant height of wt and pif7-3 plants grown under white light and shade conditions.

[0035] FIG. 5 comprises photographs of wt and mutant plants grown under various light conditions, and a plot showing flowering times of wild type and mutant plants under various light and heat conditions.

[0036] FIG. 6 shows bar plots showing the number of branches with silicles and yield of wt and mutant plants grown under white light and shade conditions.

[0037] FIG. 7A is a plot showing the effect of shade and heat on total root length in wild type and mutant pennycress plants.

[0038] FIG. 7B is a plot showing the effect of shade and heat on lateral root number in wild type and mutant pennycress plants.

[0039] FIG.7C is a plot showing the effect of shade and heat on primary root length in wild type and mutant pennycress plants.

[0040] FIG. 7D is a plot showing the effect of shade and heat on lateral root length in wild type and mutant pennycress plants. Lateral root length of plants was measured for plants grown on MS plates for 7 d then transferred to turface and grown in treatment conditions for 10 d. Roots were washed, scanned, and analyzed with Rhizovision. Statistical significance was determined by 2-tailed Welch’s t-test (± SD, n > 7, p < 0.05).

[0041] FIG. 7E is a plot showing the root:shoot ratio calculated from roots and shoots dried down after the root washing and analysis to measure biomass. Statistical significance was determined by 2-tailed Welch’s t-test (± SD, n > 7, p < 0.05). Common letters indicated no significant difference.

[0042] FIG. 8 is a plot showing the effect of ELF3 mutations on hypocotyl length in pennycress.

[0043] FIG. 9A is a bar plot showing hypocotyl length of 5 d-old seedlings of pif7-3 allele in Spring32 and tt8ARV1 genetic backgrounds. Seeds were germinated in white light for 2 d, then grown in heat for an additional 3 d. Statistical significance determined by 2- tailed Welch’s t-test (± SD, n > 19, p < 0.05). Common letters indicated no significant difference.

[0044] FIG. 9B is a PIF7 gene diagram depicting additional alleles generated by CRISPR-CAS9. Exons are black boxes. pif7-4, in the Spring32 genetic background, is a small deletion of 11 bases that results in a frameshift. pif7-5, in the tt8ARV1 background is an inversion of the gene sequence between the gRNA targets.

[0045] FIG. 9C is a bar plot showing hypocotyl length of 5 d-old seedlings, germinated in white light for 2 d, then grown in heat for an additional 3 d. Statistical significance determined by 2-tailed Welch’s t-test (± SD, n > 21 , p < 0.05). Common letters indicated no significant difference.

[0046] FIG 10A is a graph showing the effect of mutated pif7 hypocotyl length in low light intensity. Seedlings were germinated in white light at 120 pmol nr2s1for 2 d. On day 3, seedlings were grown under various neutral density filters to reduce PAR. Hypocotyls were measured on day 6. Statistical significance was determined by 2-tailed Welch’s t-test. (± SD, n > 14, p* < 0.05, p** < 0.01 , p*** <0.001 ).

[0047] FIG. 10B is a photograph of WT and mutant seedlings showing representative hypocotyls at 45 pmol rrr2s-1. Scale bar = 5 mm.

[0048] FIG. 10C is a bar plot showing hypocotyl length of seedings germinated at 120 pmol rrr2s-1for 2 d then grown under LEE209 filter to reduce PAR to 45 pmol rrr2s-1foran additional 4 d. Statistical significance was determined by 2-tailed Welch’s t-test (± SD, n > 14, p < 0.05).

[0049] FIG. 10D are photographs of representative leaves of plants grown under control light and low light conditions. Plants were sown and grown for 1 week at 300 pmol nr2s-1. Half of the plants were moved to 80 pmol rrr2s-1of light and grown for an additional week, the other half was kept under control light conditions. On day 15 plants were dissected. Leaves were arranged from oldest to youngest. Scale bar = 1 cm.

[0050] FIG. 10E is a plot showing petiole length of each leaf of wild type and pif7-3 grown at 80 pmol m-2 s-1 . Statistical significance determined by 2-tailed Welch’s t-test (± SD, n > 14, p* < 0.05, p** < 0.01 , p*** <0.001 ).

[0051] FIG. 11A is a bar plot showing Fv / Fm in wild type and pif7-3 mutants measured in dark adapted plants after 14 d of being grown in the indicated environment. Seeds were sown in soil and grown in long day control conditions (22 °C, 200 pmol nr2s-1, R:Fr =12) for 7 d. On day 7, conditions were changed to shade (22 °C, 200 pmol nr2s-1, R:Fr = 0.3), heat (28 °C, 200 pmol rm2s'1, R:Fr =12) or shade + heat (28 °C, 200 pmol rrr2s'1, R:Fr = 0.3). Elevated temperature and the far-red ratio were maintained only during the day. Statistical significance was determined by a 2-tailed Wesch’s t-test (±SD, n >16, p< 0.05). Common letters indicated no significant difference.

[0052] FIG. 11 B is a bar plot showing NPQ in wild type and pif7-3 mutants measured in dark adapted plants after 14 d of being grown in the indicated environment. Seeds were sown in soil and grown in long day control conditions (22 °C, 200 pmol nr2s-1, R:Fr =12) for 7 d. On day 7, conditions were changed to shade (22 °C, 200 pmol nr2s-1, R:Fr = 0.3), heat (28 °C, 200 pmol nr2s'1, R:Fr =12) or shade + heat (28 °C, 200 pmol rrr2s-1, R:Fr = 0.3). Elevated temperature and the far-red ratio were maintained only during the day. Statistical significance was determined by a 2-tailed Wesch’s t-test (±SD, n >16, p< 0.05). Common letters indicated no significant difference.

[0053] FIG. 11C is a plot showing NDVI in wild type and pif7-3 mutants measured in dark adapted plants after 14 d of being grown in the indicated environment. Seeds were sown in soil and grown in long day control conditions (22 °C, 200 pmol nr2s-1, R:Fr =12) for 7 d. On day 7, conditions were changed to shade (22 °C, 200 pmol nr2s-1, R:Fr =0.3), heat (28 °C, 200 pmol nr2s'1, R:Fr =12) or shade + heat (28 °C, 200 pmol nr2s-1, R:Fr = 0.3). Elevated temperature and the far-red ratio were maintained only during the day. Statistical significance was determined by a 2-tailed Wesch’s t-test (±SD, n >16, p< 0.05). Common letters indicated no significant difference.

[0054] FIG. 11 D is a plot showing CIRE in wild type and pif7-3 mutants measured in dark adapted plants after 14 d of being grown in the indicated environment. Seeds were sown in soil and grown in long day control conditions (22 °C, 200 pmol rm2s-1, R:Fr =12) for 7 d. On day 7, conditions were changed to shade (22 °C, 200 pmol rm2s-1, R:Fr = 0.3), heat (28 °C, 200 pmol nr2s'1, R:Fr =12) or shade + heat (28 °C, 200 pmol nr2s-1, R:Fr = 0.3). Elevated temperature and the far-red ratio were maintained only during the day. Statistical significance was determined by a 2-tailed Wesch’s t-test (±SD, n >16, p< 0.05). Common letters indicated no significant difference.

[0055] FIG. 12A is a plot showing percent green coverage within the boundary of the mini-field was calculated from overheard images taken every 5 days post vernalization with PlantCV.

[0056] FIG. 12B is a bar plot showing solidity measurements from 5 rosettes pulled at random from each mini field 11 d after vernalization. Statistical significance determined by 2-tailed Mann-Whitney test (±SD, n = 15, p < 0.05). Common letters indicated no significant difference.

[0057] FIG. 12C is a bar plot showing convex hull area of the 5 rosettes of FIG. 11 B, calculated using the morphology function from PlantCV. Statistical significance determined by 2-tailed Mann-Whitney test (±SD, n = 15, p < 0.05). Common letters indicated no significant difference.

[0058] FIG. 12D are photographs of mini-fields 40 d after vernalization.

[0059] FIG. 12E is a plot quantifying Inflorescences with open flowers counted in each mini-field.

[0060] FIG. 12F is a bar plot showing average yield from all seeds harvested for each mini-field. Statistical significance determined by 2-tailed Mann-Whitney test (±SD, n = 3, p < 0.05). Common letters indicated no significant difference.DETAILED DESCRIPTION

[0061] The present disclosure is based in part on the discovery that reducing the expression of light signaling pathways in pennycress results in plants that show reduced responses to shade, including foliar shade, and elevated temperatures. More specifically, the inventors generated genetically modified pennycress plants comprising mutations that reduce the expression of PIF7 and established that the genetically modified pennycress plants exhibit reduced elongation responses and enhanced their suitability for high-density planting. The genetically modified plants exhibit a shorter time to canopy closure, enhanced weed suppression, increased biomass, and maintain similar yields compared to wild-type plants. By addressing the elongation issue, these modified pennycress plants offer greater vigor and productivity, making them more robust and effective for integration into existing agricultural systems.I. Genetically modified pennycress plant

[0062] One aspect of the instant disclosure encompasses a genetically modified pennycress plant with improved response to shade and elevated temperatures. The genetically modified pennycress plant or part thereof, plant cell, or seed comprises a genetic modification of at least one target site that modifies expression of a PIF7 protein.

[0063] Importantly, the inventors surprisingly discovered that mutating PIF7 in pennycress results in plants that exhibit shorter time to canopy closure, enhanced weed suppression, increased biomass at high planting densities, and higher yield. This is particularly advantageous in scenarios where pennycress is inter-seeded with corn, as the modified plants can be planted in late fall into standing corn fields. By lowering elongation responses to shade and elevated temperature, pennycress ground cover can be increased when grown at high densities, reducing foliar shade-induced responses when inter-seeded into standing crops, and limiting elongation in response to higher temperatures. This allows for more efficient land use during the fallow period between corn harvest and the next planting season. Overall, plants comprising the edited PIF7gene have higher vigor than wild type, making them well-suited for integration into existing agricultural systems alongside crops like corn.(a) Pennycress

[0064] Pennycress, scientifically known as Thlaspi arvense and commonly referred to as field pennycress, is a member of the Brassicaceae family, which includes other well- known plants such as mustard, cabbage, and broccoli. This plant is under development as a new winter annual bioenergy oilseed to be grown as a third crop in the Midwest. Pennycress doesn’t compete with current crops as it is planted outside of the regular growing season. It is planted in winter and harvested in the spring due to its exceptional cold tolerance. Its seeds are rich in oil (35%) and protein (27%), making it a valuable resource for biofuel production. In addition, planting pennycress outside the growing season protects soil from erosion with broad rosettes, prevents pollution by removing excess fertilizer from soil, and reduces winter annual weed pressure, contributing to environmental sustainability.

[0065] Pennycress is most closely related to other species within the genus Thlaspi, such as Thlaspi perfoliatum and Thlaspi caerulescens. While these species are not widely cultivated as crop plants, they share several traits with pennycress, including adaptability to various climates and habitats. Thlaspi caerulescens, for instance, is valued for its exceptional ability to hyperaccumulate heavy metals, making it a key species in phytoremediation research. Similarly, Thlaspi perfoliatum demonstrates resilience in diverse environments, though it lacks the bioenergy or agronomic traits necessary for large-scale agricultural use.

[0066] The genus Thlaspi itself is closely related to other genera in the Brassicaceae family, such as Arabidopsis, Brassica, and Camelina. Among these, Camelina sativa (false flax) stands out as a close agricultural counterpart to pennycress. Like pennycress, Camelina is a cold-tolerant oilseed crop, widely grown for biofuel production. Additionally, Arabidopsis thaliana serves as a key model organism for genetic and molecular studies, offering insights into conserved genetic pathways within the Brassicaceae family.

[0067] Pennycress, as both a wild plant and an emerging crop, occupies a unique niche within this family. Its close relationship with other Brassicaceae members highlights its evolutionary significance and its potential as a winter annual optimized for bioenergy production and environmental sustainability.

[0068] As detailed above, the inventors have demonstrated that mutating the PIF7 gene in pennycress results in plants with an enhanced ability to respond to shade and elevated temperatures such as in situations where pennycress is inter-seeded with com. Wild-type pennycress plants typically struggle under these conditions, exhibiting intolerance to both shade and heat. In contrast, the genetically modified pennycress plants described in the instant disclosure are shorter when grown in shaded environments and at higher temperatures. This reduced elongation is advantageous for cultivation, as it leads to a shorter time to canopy closure, which enhances weed suppression and increases biomass when planted at high densities. Furthermore, these modified plants maintain similar yields under shade and elevated temperature conditions compared to their wild-type counterparts. By minimizing elongation responses, the genetically modified pennycress can provide better ground cover, reduce shade-induced responses when inter-seeded with standing crops, and limit elongation due to higher temperatures. Overall, the edited PIF7 gene confers greater vigor to the plants, making them more robust and productive in challenging growing conditions.

[0069] In some aspects, the genetically modified pennycress plant or part thereof, plant cell, or seed of the instant disclosure is a Thlaspi species. In some aspects, the genetically modified pennycress plant or part thereof, plant cell, or seed of the instant disclosure is Thlaspi perfoliatum. In some aspects, the genetically modified pennycress plant or part thereof, plant cell, or seed of the instant disclosure is Thlaspi caerulescens. In some aspects, the genetically modified pennycress plant or part thereof, plant cell, or seed of the instant disclosure is Thlaspi arvense.(b) PIF7

[0070] The PHYTOCHROME-INTERACTING FACTOR (PIF) genes encode a family of basic helix-loop-helix (bHLH) transcription factors that are highly conserved across plant species and are integral to plant growth and development. PIFs play key roles in modulating gene expression in response to environmental cues such as light and temperature. In many plants, PIFs interact with light-sensing phytochromes to regulate processes including seed germination, hypocotyl elongation, and shade avoidance. These genes are also critical for integrating temperature signals, influencing thermomorphogenesis by modulating hormonal pathways like auxin biosynthesis.

[0071] The PIF family consists of several genes, including PIF7, PIF4, PIF5, and PIF3, each of which contributes to plant responses to environmental cues. PIF7 integrates light and temperature signals and regulates growth responses, including thermomorphogenesis and shade avoidance. PIF4 and PIF5 are closely related to PIF7 and also contribute to thermomorphogenesis and shade avoidance, sharing overlapping roles in integrating temperature signals while maintaining specific roles in particular light responses. In contrast, PIF3 is more specialized in light signaling and acts as an early responder in the phytochrome pathway, with less involvement in temperature-related responses compared to PIF7.

[0072] In Brassica plants, the PIF gene family shows functional conservation with additional adaptations that allow for fine-tuned responses to specific agricultural environments. For example, PIFs in Brassica regulate flowering time and stress tolerance, traits crucial for crop yield and resilience. Studies suggest functional divergence among Brassica PIFs, with certain homologs displaying enhanced roles in shade avoidance and temperature adaptation compared to other plant species.

[0073] In pennycress (Thlaspi arvense), a Brassica-relative and an emerging model for cover crops, PIF genes exhibit unique regulatory patterns. These genes contribute to its robust adaptability to various environments, including its ability to thrive in cooler temperatures. Research indicates that PIF7, in particular, integrates light and temperature signals to modulate developmental transitions, such as flowering and biomass accumulation, optimizing growth for challenging climates.

[0074] Importantly, the inventors surprisingly discovered that pennycress plants genetically modified to alter the expression of PIF7 produces plants with reduced elongation when grown at high planting density compared to similarly grown wild type plants. Plants comprising genetically edited PIF7 gene also have reduced elongation at elevated temperatures. This is a surprising result because the response of pennycress to the combination of shade and heat is strikingly different from the response seen in related plants such as Arabidopsis. For instance, in Arabidopsis, the response to shade and heat are additive, whereas the response to shade and heat in pennycress is epistatic. Further, although the inventors discovered that mutating PIF7 in pennycress alters the response of the plant to shade and heat, mutating other components of the light signaling pathways that have been shown to be effective in other related plants were not effective in pennycress.

[0075] Accordingly, in some aspects, a genetically modified Thalspi sp. plant or part thereof, plant cell, or seed of the instant disclosure comprises a genetic modification of at least one target site, wherein the genetic modification modifies expression of a PIF7 protein. In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 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% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0076] In some aspects, the genetically modified Thalspi sp. plant is a loss of function mutation. In some aspects, the genetic modification is a loss of function mutation in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetic modification is a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein.

[0077] In some aspects, the endogenous nucleic acid sequence encoding the PIF7 protein comprising the loss of function mutation comprises at least about 75% or more,at least about 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% sequence identity with a nucleic acid sequence of SEQ ID NO: 3 (pif7-3), SEQ ID NO: 4 (pif7-1), SEQ ID NO: 5 (p / 77-2), SEQ ID NO: 6 (pif7-4), and SEQ ID NO: 7 (pif7-5). In some aspects, the endogenous nucleic acid sequence encoding the PIF7 protein comprising the loss of function mutation comprises at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0078] The modification of the polynucleotide encoding the PIF7 protein can comprise a modification introduced by a programmable nucleic acid modification system comprising a CRISPR / Cas nuclease system, wherein the CRISPR / Cas nuclease system comprises a CAS9 protein (CAS9) and a guide RNA (gRNA).

[0079] In some aspects, the Cas9 nuclease is encoded by a nucleic acid sequence comprising about 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% sequence identity with the nucleic acid sequence of SEQ ID NO: 8. In some aspects, the Cas9 nuclease is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 8.

[0080] In some aspects, the gRNA comprises a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof.(c) Aspects

[0081] In some aspects, a genetically modified plant of the instant disclosure genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response to shade and higher temperatures. The genetically modified plant comprises a genetic modification of at least one target site, wherein the genetic modification modifies expression of a PIF7 protein. The genetic modification confers an improved response to shade and higher temperatures to the Thlaspi sp. plant.

[0082] In some aspects, when the , the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 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% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0083] In some aspects, the genetic modification is in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetic modification is a loss of function mutation. The genetic modification can be in an endogenous nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetic modification is a loss of function mutation that reduces or eliminates the expression of the PIF7 protein. In some aspects, the genetic modification is a loss of function mutation in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetic modification is a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein.

[0084] The Thalspi sp. plant can be Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense. For instance, the Thalspi sp. plant can be Thlaspi arvense (pennycress). In some aspects, the genetically modified plant of the instant disclosure is a Thlaspi arvense plant. In some aspects, the genetically modified plant is a Thlaspi arvense plant comprising a genetic modification in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetically modified plant is a Thlaspi arvense plant comprising a genetic modification in an endogenous nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetically modified plant is a Thlaspi arvense plant comprising a loss of function mutation in a nucleic acid sequence encoding the PIF7 protein. In some aspects, the genetically modified plant is a Thlaspi arvense plant comprising a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein.

[0085] In some aspects, the genetically modified plant is a Thlaspi arvense plant comprising a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein, wherein the endogenous nucleic acid sequence encoding the PIF7 protein comprising the loss of function mutation comprises at least about 75% or more, at least about 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% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: X, and SEQ ID NO: Y. In some aspects, the endogenous nucleic acid sequence encoding the PIF7 protein comprising the loss of function mutation comprises at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: X, and SEQ ID NO: Y.II. Engineered nucleic acid modification system

[0086] Another aspect of the present disclosure encompasses an engineered nucleic acid modification system for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed. The engineered nucleic acid modification system introduces a genetic modification of at least one target site. The genetic modification modifies expression of a PIF7 protein in a Thlaspi sp. plant or part thereof, plant cell, or seed. Non-limiting examples of suitable protein expression modification systems include programmable nucleic acid modification systems, an expression construct encoding a protein or variants thereof, and any combination thereof.

[0087] In some aspects, the nucleic acid modification system is an expression construct comprising a nucleotide sequence encoding the polypeptide or polynucleotide operably linked to a promoter. In other aspects, the nucleic acid modification system is a programmable nucleic acid modification system targeted to a nucleic acid sequence in a nucleotide sequence encoding the PIF7 protein. As used herein, a “programmable nucleic acid modification system” is a system capable of targeting and modifying the nucleic acid or modifying the expression or stability of a nucleic acid to alter apolynucleotide sequence or a protein or the expression of a polynucleotide sequence or protein encoded by the nucleic acid. The programmable nucleic acid modification system can comprise an interfering nucleic acid molecule or a nucleic acid editing system. The programmable protein expression modification system can be specifically targeted to a target site within a nucleic acid sequence encoding a polypeptide or a polynucleotide a PIF7 protein.

[0088] In some aspects, the programmable expression modification system comprises an interfering nucleic acid (RNAi) molecule having a nucleotide sequence complementary to a target sequence within a gene encoding the polypeptide or polynucleotide used to inhibit expression of the polypeptide or polynucleotide. RNAi molecules generally act by forming a heteroduplex with a target RNA molecule, which is selectively degraded or “knocked down,” hence inactivating the target RNA. Under some conditions, an interfering RNA molecule can also inactivate a target transcript by repressing transcript translation and / or inhibiting transcription. An interfering RNA is more generally said to be “targeted against” a biologically relevant target, such as a protein, when it is targeted against the nucleic acid encoding the target. For example, an interfering RNA molecule has a nucleotide (nt) sequence which is complementary to an endogenous mRNA of a target gene sequence. Thus, given a target gene sequence, an interfering RNA molecule can be prepared which has a nucleotide sequence at least a portion of which is complementary to a target gene sequence. When introduced into cells, the interfering RNA binds to the target mRNA, thereby functionally inactivating the target mRNA and / or leading to degradation of the target mRNA.

[0089] Interfering RNA molecules include, inter alia, small interfering RNA (siRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), long non-coding RNAs (long ncRNAs or IncRNAs), and small hairpin RNAs (shRNA). IncRNAs are widely expressed and have key roles in gene regulation. Depending on their localization and their specific interactions with DNA, RNA and proteins, IncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs, and interfere with signaling pathways. Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs regulate gene expression through interactions with piwi-subfamily Argonaute proteins. SiRNA are double-stranded RNA molecules, preferably about 19-25 nucleotides in length. When transfected into cells, siRNA inhibit the target mRNA transiently until they are also degraded within the cell. MiRNA and siRNA are biochemically and functionally indistinguishable. Both are about the same in nucleotide length with 5’-phosphate and 3’-hydroxyl ends, and assemble into an RNA- induced silencing complex (RISC) to silence specific gene expression. siRNA and miRNA are distinguished based on origin. siRNA is obtained from long double-stranded RNA (dsRNA), while miRNA is derived from the double-stranded region of a 60-70nt RNA hairpin precursor. Small hairpin RNAs (shRNA) are sequences of RNA, typically about 50-80 base pairs, or about 50, 55, 60, 65, 70, 75, or about 80 base pairs in length, that include a region of internal hybridization forming a stem loop structure consisting of a base-pair region of about 19-29 base pairs of double-strand RNA (the stem) bridged by a region of single-strand RNA (the loop) and a short 3’ overhang. shRNA molecules are processed within the cell to form siRNA which in turn knock down target gene expression. shRNA can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression, and thus longer knockdown of the target mRNA.

[0090] Interfering nucleic acid molecules can contain RNA bases, non-RNA bases, or a mixture of RNA bases and non-RNA bases. For example, interfering nucleic acid molecules provided herein can be primarily composed of RNA bases but also contain DNA bases or non-naturally occurring nucleotides. The interfering nucleic acids can employ a variety of oligonucleotide chemistries. Examples of oligonucleotide chemistries include, without limitation, peptide nucleic acid (PNA), linked nucleic acid (LNA), phosphorothioate, 2'0-Me-modified oligonucleotides, and morpholino chemistries, including combinations of any of the foregoing. In general, PNA and LNA chemistries can utilize shorter targeting sequences because of their relatively high target binding strength relative to 2'0-Me oligonucleotides. Phosphorothioate and 2'0-Me-modified chemistries are often combined to generate 2'0-Me-modified oligonucleotides having a phosphorothioate backbone.

[0091] In some aspects, the programmable nucleic acid modification system is a nucleic acid editing system. Such modification system can be used to edit DNA or RNA sequences to repress transcription or translation of an mRNA encoded by the gene, and / or produce mutant proteins with reduced activity or stability. Non-limiting examples of programmable nucleic acid editing systems include, without limit, an RNA-guided clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR- associated (Cas) (CRISPR / Cas) nuclease system, a CRISPR / Cpf1 nuclease system, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a ribozyme, or a programmable DNA binding domain linked to a nuclease domain. Other suitable programmable nucleic acid modification systems will be recognized by individuals skilled in the art.

[0092] Such systems rely for specificity on the delivery of exogenous protein(s), and / or a guide RNA (gRNA) or single guide RNA (sgRNA) having a sequence which binds specifically to a gene sequence of interest. When the programmable nucleic acid modification system comprises more than one component, such as a protein and a guide nucleic acid, the multi-component modification system can be modular, in that the different components can optionally be distributed among two or more nucleic acid constructs as described herein. The system components can be delivered by a plasmid or viral vector or as a synthetic oligonucleotide. More detailed descriptions of programmable nucleic acid editing systems can be as described further below.

[0093] In some aspects, the programmable nucleic acid modification system is a CRISPR / Cas tool modified for transcriptional regulation of a locus. In some aspects, the programmable nucleic acid modification system is CRISPR / Cas system comprising a Cas9 nuclease and a guide RNA (gRNA) comprising a sequence complementary to a target sequence within the nucleotide sequence encoding PIF7.

[0094] In some aspects, the Cas9 nuclease is encoded by a nucleic acid sequence comprising about 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%sequence identity with the nucleic acid sequence of SEQ ID NO: 8. In some aspects, the Cas9 nuclease a nucleic acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 8.

[0095] In some aspects, the genetically modified plant is Thlaspi arvense. In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising about 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% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. When the programmable nucleic acid modification system is a CRISPR / Cas system, the gRNA can comprise a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or any combination thereof.

[0096] Another aspect of the instant disclosure encompasses a Thlaspi sp. plant or plant cells comprising an engineered nucleic acid modification system for introducing a genetic modification of at least one target site. The engineered nucleic acid modification system can be as described herein above. / . CRISPR nuclease systems.

[0097] The programmable targeting nuclease can be an RNA-guided CRISPR endonuclease system. The CRISPR system comprises a guide RNA or sgRNA to a target sequence at which a protein of the system introduces a double-stranded break in a target nucleic acid sequence, and a CRISPR-associated endonuclease. The gRNA is a short synthetic RNA comprising a sequence necessary for endonuclease binding, and a preselected ~20 nucleotide spacer sequence targeting the sequence of interest in a genomic target. Non-limiting examples of endonucleases include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1 , Csy2, Csy3, Cse1 , Cse2, Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, or Cpf1 endonuclease, or a homolog thereof, a recombination of the naturally occurring molecule thereof, a codon- optimized version thereof, or a modified version thereof, or any combination thereof. Additional programmable nucleic acid modification systems include the CRISPR-Cas12 and CRISPR-Cas13 systems, which target single-stranded DNA and RNA, respectively. CRISPR-Cas12 systems, such as Cas12a (Cpf1 ), Cas12b, and their engineered variants, are capable of both site-specific and collateral cleavage of single-stranded DNA. CRISPR-Cas13 systems, such as Cas13a, Cas13b, and Cas13d, utilize RNA- guided targeting to cleave single-stranded RNA, with applications in transcriptome editing and RNA-based diagnostics.

[0098] The CRISPR nuclease system can be derived from any type of CRISPR system, including a type I (i.e. , IA, IB, IC, ID, IE, or IF), type II (i.e. , HA, IIB, or IIC), type III (i.e. , 11 IA or 11 IB), or type V CRISPR system. The CRISPR / Cas system can be from Streptococcus sp. (e.g., Streptococcus pyogenes), Campylobacter sp. e.g., Campylobacter jejuni), Francisella sp. (e.g., Francisella novicida), Acaryochloris sp., Acetohalobium sp., Acidami nococcus sp., Acidithiobacillus sp., Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp., Ktedonobacter sp., Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscilla sp., Microcoleus sp., Microcystis sp., Natranaerobius sp., Neisseria sp., Nitrosococcus sp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oscillatorla sp., Polaromonas sp., Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp., Streptomyces sp., Streptosporangium sp., Synechococcus sp., or Thermosipho sp.

[0099] Non-limiting examples of suitable CRISPR systems include CRISPR / Cas systems, CRISPR / Cpf systems, CRISP R / Cmr systems, CRISPR / Csa systems, CRISPR / Csb systems, CRISPR / Csc systems, CRISPR / Cse systems, CRISPR / Csf systems, CRISPR / Csm systems, CRISPR / Csn systems, CRISPR / Csx systems,CRISPR / Csy systems, CRISPR / Csz systems, CRISPR / Cas12 systems, CRISPR / Cas13 systems, and derivatives or variants thereof. Preferably, the CRISPR system can be a type II Cas9 protein, a type V Cpf1 protein, a type V Cas12 protein, a type VI Cas13 protein, or a derivative thereof. In some aspects, the CRISPR / Cas nuclease is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), Francisella novicida Cpf1 (FnCpfl ), Lachnospiraceae bacterium Cas12a, or Leptotrichia wadei Cas13a.

[0100] In general, a protein of the CRISPR system comprises an RNA recognition and / or RNA binding domain, which interacts with the guide RNA. A protein of the CRISPR system also comprises at least one nuclease domain having endonuclease activity. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and an HNH-like nuclease domain, while a Cpf1 protein (now more commonly referred to as Cas12a) can comprise a RuvC-like domain. Beyond these, CRISPR systems have expanded to include additional proteins, such as Cas12b, Cas13, and Cas1 , each with distinct properties and functions. Cas13 proteins, for example, exhibit RNA-targeting activity and lack DNA endonuclease activity, while Cas14 proteins are notable for their ability to target single-stranded DNA with high specificity. A protein of the CRISPR system can also comprise DNA binding domains, helicase domains, RNase domains, protein-protein interaction domains, dimerization domains, as well as other domains. Furthermore, advancements have identified engineered variants of Cas9 and other Cas proteins, such as high-fidelity Cas9 (HF-Cas9) and Cas9 nickase, which offer improved specificity and reduced off-target effects.

[0101] A protein of the CRISPR system can be associated with guide RNAs (gRNA). The guide RNA can be a single guide RNA (i.e. , sgRNA), or can comprise two RNA molecules (i.e., crRNA and tracrRNA). The guide RNA interacts with a protein of the CRISPR system to guide it to a target site in the DNA. The target site generally has no sequence limitation except that the sequence is bordered by a protospacer adjacent motif (PAM). For example, PAM sequences for Cas9 include 3'-NGG, 3'-NGGNG, 3'- NNAGAAW, and 3'-ACAY, and PAM sequences for Cpf1 (now referred to as Cas12a)include 5'-TTN (wherein N is defined as any nucleotide, W is defined as either A or T, and Y is defined as either C or T). Expanding knowledge of CRISPR systems has revealed diverse PAM sequences for additional CRISPR-associated proteins, such as Cas12b, Cas12f, and Cas14, enabling targeting flexibility. Each gRNA comprises a sequence that is complementary to the target sequence (e.g., a Cas9 gRNA can comprise GN17-20GG). The gRNA can also comprise a scaffold sequence that forms a stem loop structure and a single-stranded region. Variations in scaffold sequences have been engineered to improve stability, binding affinity, and target specificity, such as incorporating secondary structure modifications or chemically modified bases. The scaffold region can be the same in every gRNA, although customizable scaffolds have been developed for enhanced functionality. In some aspects, the gRNA can be a single molecule (i.e. , sgRNA), and in other aspects, the gRNA can be two separate molecules. Furthermore, advances in gRNA design tools, including machine learning-based algorithms and high-throughput screening, provide researchers with improved prediction of on-target and off-target activity. These gRNA design tools are available on the internet or from commercial sources.

[0102] A CRISPR system can comprise one or more nucleic acid binding domains associated with one or more, or two or more selected guide RNAs used to direct the CRISPR system to one or more, or two or more selected target nucleic acid loci. For instance, a nucleic acid binding domain can be associated with one or more, or two or more selected guide RNAs, each selected guide RNA, when complexed with a nucleic acid binding domain, causing the CRISPR system to localize to the target of the guide RNA. Recent advancements in CRISPR systems have enabled the use of multiplexed guide RNA strategies, where multiple guide RNAs are simultaneously delivered to target different loci within the same system, facilitating complex genome editing tasks, such as large-scale deletions, insertions, or gene regulation. Additionally, engineered or modified nucleic acid binding domains, such as deactivated Cas9 (dCas9), have been developed to enable precise targeting without cleavage, expanding applications to include transcriptional regulation, epigenetic editing, and imaging of genomic loci. Modular fusion proteins combining CRISPR-associated nucleic acidbinding domains with effector proteins, such as transcriptional activators, repressors, or base editors, have also been widely adopted, further diversifying the capabilities of CRISPR systems. / / . CRISPR nickase systems.

[0103] The programmable targeting nuclease can also be a CRISPR nickase system. CRISPR nickase systems are similar to the CRISPR nuclease systems described above except that a CRISPR nuclease of the system is modified to cleave only one strand of a double-stranded nucleic acid sequence. Thus, a CRISPR nickase, in combination with a guide RNA of the system, can create a single-stranded break or nick in the target nucleic acid sequence. Alternatively, a CRISPR nickase in combination with a pair of offset gRNAs can create a double-stranded break in the nucleic acid sequence. Recent advancements have highlighted the utility of CRISPR nickase systems for high-precision genome editing applications, such as reducing off- target effects and enhancing the accuracy of homology-directed repair (HDR) or prime editing. Furthermore, nickase systems are increasingly being used in base-editing platforms, where they catalyze precise nucleotide conversions without inducing doublestranded breaks.A CRISPR nuclease of the system can be converted to a nickase by one or more mutations and / or deletions. For example, a Cas9 nickase can comprise one or more mutations in one of the nuclease domains, wherein the one or more mutations can be D10A, E762A, and / or D986A in the RuvC-like domain, or the one or more mutations can be H840A (or H839A), N854A, and / or N863A in the HNH-like domain. Engineered nickase systems have also been adapted for novel functions, such as precise transcriptional modulation or epigenetic editing when fused with effectors like deaminases or methyltransferases. These innovations significantly expand the versatility of CRISPR nickase systems in both research and therapeutic applications.Hi. ssDNA -guided A rgonaute systems.

[0104] Alternatively, the programmable targeting nuclease can comprise a singlestranded DNA-guided Argonaute (AGO) endonuclease. Argonaute (AGO) proteins are a family of endonucleases that use 5'-phosphorylated short single-stranded nucleic acids as guides to cleave nucleic acid targets. Some prokaryotic AGO proteins use singlestranded guide DNAs (gDNAs) and create double-stranded breaks in nucleic acid sequences. Recent studies have expanded the potential applications of AGO endonucleases to include precise genome editing, gene silencing, and antiviral defenses. Unlike CRISPR systems, AGO proteins do not require a protospacer adjacent motif (PAM), making them versatile tools for targeting a broader range of sequences. The ssDNA-guided AGO endonuclease can be associated with a single-stranded guide DNA (gDNA) and has demonstrated promise in diverse model systems.

[0105] The AGO endonuclease can be derived from Alistipes sp., Aquifex sp., Archaeog / obus sp. , Bacteroides sp. , Bradyrhizobium sp. , Burkholderia sp. , Cellvibrio sp., Chlorobium sp., Geobactersp., Mari profundus sp., Natronobacterium sp., Parabacteroldes sp., Parvularcula sp., Planctomyces sp., Pseudomonas sp., Pyrococcus sp., Thermus sp., or Xanthomonas sp. For instance, the AGO endonuclease can be Natronobacterium gregoryi AGO (NgAGO), Thermus thermophilus AGO (TtAGO), or Pyrococcus furiosus AGO (PfAGO). Recent engineering efforts have further improved AGO functionality, such as enhancing its activity in eukaryotic cells, which has traditionally been a limitation for some prokaryotic AGO proteins. Modified versions of TtAGO and NgAGO have shown increased stability and targeting efficiency, broadening their use in genome manipulation and diagnostics.

[0106] The single-stranded guide DNA (gDNA) of an ssDNA-guided Argonaute system is complementary to the target site in the nucleic acid sequence. The target site has no sequence limitations and does not require a PAM, offering significant flexibility in target selection compared to CRISPR systems. The gDNA generally ranges in length from about 15-30 nucleotides and typically comprises a 5' phosphate group for efficient binding and activity. Advances in oligonucleotide chemistry have introduced modifications, such as phosphorothioate linkages or locked nucleic acids, to improve the stability and specificity of gDNAs in AGO systems. Those skilled in the art arefamiliar with ssDNA oligonucleotide design and construction, with design tools and high- throughput synthesis platforms readily available for customization. Additionally, applications of AGO systems in diagnostic tools, such as nucleic acid detection and genotyping, are being actively developed. iv. Zinc finger nucleases.

[0107] The programmable targeting nuclease can be a zinc finger nuclease (ZFN). A ZFN comprises a DNA-binding zinc finger region and a nuclease domain. The zinc finger region can comprise from about two to seven zinc fingers, for example, about four to six zinc fingers, wherein each zinc finger binds three nucleotides. The zinc finger region can be engineered to recognize and bind to any DNA sequence. Recent developments in computational tools and high-throughput screening technologies have enhanced the precision and efficiency of zinc finger design. Zinc finger design tools or algorithms are available on the internet or from commercial sources, enabling the rapid customization of ZFNs for diverse genomic targets. The zinc fingers can be linked together using suitable linker sequences, which have also been optimized in recent years to improve flexibility, stability, and binding efficiency.

[0108] A ZFN also comprises a nuclease domain, which can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a nuclease domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. The nuclease domain can be derived from a type I l-S restriction endonuclease. Type ll-S endonucleases cleave DNA at sites that are typically several base pairs away from the recognition / binding site and, as such, have separable binding and cleavage domains. These enzymes generally are monomers that transiently associate to form dimers to cleave each strand of DNA at staggered locations. Non-limiting examples of suitable type ll-S endonucleases include Bfil, Bpml, Bsal, Bsgl, BsmBI, Bsml, BspMI, Fokl, Mboll, and Sapl.

[0109] The type ll-S nuclease domain can be modified to facilitate dimerization of two different nuclease domains. Advances in ZFN engineering have focused onreducing off-target effects and enhancing dimerization specificity. For example, the cleavage domain of Fokl can be modified by mutating certain amino acid residues. By way of non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491 , 496, 498, 499, 500, 531 , 534, 537, and 538 of Fokl nuclease domains are targets for modification. Specific modifications, such as Q486E, I499L, and / or N496D mutations in one Fokl domain and E490K, I538K, and / or H537R mutations in the other, enhance heterodimerization and reduce homodimerization, thus minimizing unintended DNA cleavage. These improvements have expanded the applications of ZFNs in both research and clinical settings, including therapeutic gene editing and functional genomic studies. v. Transcription activator-like effector nuclease systems.

[0110] The programmable targeting nuclease can also be a transcription activator-like effector nuclease (TALEN) or the like. TALENs comprise a DNA-binding domain composed of highly conserved repeats derived from transcription activator-like effectors (TALEs) that are linked to a nuclease domain. TALEs are proteins secreted by plant pathogen Xanthomonas to alter transcription of genes in host plant cells. TALE repeat arrays can be engineered via modular protein design to target any DNA sequence of interest. Recent advancements have improved the efficiency and specificity of TALENs through optimized repeat arrays, linker sequences, and nuclease domain modifications. TALENs have also been increasingly utilized in applications requiring high precision, such as therapeutic gene editing and somatic cell genome engineering, owing to their minimal off-target effects compared to some other programmable nucleases.

[0111] Other transcription activator-like effector nuclease systems can comprise, but are not limited to, the repetitive sequence, transcription activator-like effector (RipTAL) system from the bacterial plant pathogenic Ralstonia solanacearum species complex (Rssc). Advances in the RipTAL system and similar technologies have expanded their utility in targeting broader genomic contexts, including sequences withcomplex secondary structures or repetitive elements. Additionally, TALEN engineering has integrated innovations such as fusion with base editors or epigenetic modifiers, further enhancing their potential for precise genetic and epigenetic modifications.

[0112] The nuclease domain of TALEs can be any nuclease domain as described above in Section ll(i). vi. Meganucleases or rare-cutting endonuclease systems.

[0113] The programmable targeting nuclease can also be a meganuclease or derivative thereof. Meganucleases are endodeoxyribonucleases characterized by long recognition sequences, generally ranging from about 12 base pairs to about 45 base pairs. As a consequence of this requirement, the recognition sequence typically occurs only once in any given genome, making meganucleases highly specific tools for genome editing. Among meganucleases, the family of homing endonucleases named LAGLIDADG has become a valuable tool for genome study and engineering. Nonlimiting examples of meganucleases that can be suitable for the instant disclosure include l-Scel, l-Crel, l-Dmol, or variants and combinations thereof. Recent advancements have focused on improving the targeting flexibility of meganucleases by engineering their DNA recognition domains, allowing them to target sequences beyond their native recognition motifs. Directed evolution and computational protein design have also been employed to develop custom meganucleases tailored for specific genomic loci with reduced off-target effects, expanding their applicability in therapeutic and research contexts.

[0114] The programmable targeting nuclease can also be a rare-cutting endonuclease or derivative thereof. Rare-cutting endonucleases are site-specific endonucleases whose recognition sequence occurs infrequently in a genome, often only once in a genome. The rare-cutting endonuclease can recognize a 7-nucleotide sequence, an 8-nucleotide sequence, or longer recognition sequence. Non-limiting examples of rare-cutting endonucleases include Notl, Asci, Pad, AsiSI, Sbfl, and Fsel. Recent advancements in rare-cutting endonucleases include their integration intosynthetic biology workflows, where they are used for precise genomic rearrangements or construction of artificial genomes. Engineered variants of rare-cutting endonucleases have also been developed to enhance activity, stability, and sequence specificity, enabling their use in complex genome engineering projects and therapeutic applications. vii. Optional additional domains.

[0115] The programmable targeting nuclease can further comprise at least one nuclear localization signal (NLS), at least one cell-penetrating domain, at least one reporter domain, and / or at least one linker.

[0116] In general, an NLS comprises a stretch of basic amino acids. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101 -5105). An NLS can be located at the N-terminus, the C-terminus, or an internal location of the fusion protein. An NLS can also be a bipartite NLS sequences that improves nuclear import efficiency, particularly in larger or complex programmable nucleases, such as Cas9 or TALENs. The strategic placement and optimization of NLS sequences have been key for efficient delivery of nucleases in both research and therapeutic applications.

[0117] A cell-penetrating domain can be a cell-penetrating peptide sequence derived from the HIV-1 TAT protein. The cell-penetrating domain can be located at the N-terminus, the C-terminus, or an internal location of the fusion protein. Additionally, alternative cell-penetrating peptides, such as those derived from arginine-rich sequences or synthetic peptides like R9, have been developed to enhance cell permeability and minimize cytotoxicity. These domains are particularly relevant in the delivery of genome-editing tools directly into cells without the need for viral or lipid- based delivery systems.

[0118] A programmable targeting nuclease can further comprise at least one linker. For example, the programmable targeting nuclease, the nuclease domain of the targeting nuclease, and other optional domains can be linked via one or more linkers.The linker can be flexible (e.g., comprising small, non-polar (e.g., Gly) or polar (e.g., Ser, Thr) amino acids). Linkers can be tunable linkers that provide optimal spacing, stability, and activity between functional domains of programmable nucleases. Examples of suitable linkers are well known in the art, and programs to design linkers are readily available (see Crasto et al., Protein Eng., 2000, 13(5):3096-312). In alternate aspects, the programmable targeting nuclease, the cell cycle-regulated protein, and other optional domains can be linked directly, allowing covalent or dynamic linkages for controlled activation or modular assembly.

[0119] A programmable targeting nuclease can further comprise an organelle localization or targeting signal that directs a molecule to a specific organelle. A signal can be a polynucleotide or polypeptide signal or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired organelle. Organelle localization signals, such as mitochondrial targeting sequences, chloroplast transit peptides, or peroxisomal targeting signals, have been refined for specific and efficient delivery to organelles. These signals are increasingly used in applications requiring subcellular genome editing or protein localization for functional studies. Organelle localization signals can be as described in U.S. Patent Publication No. 20070196334, the disclosure of which is incorporated herein in its entirety. Recent advances have also explored programmable localization to organelles for targeted editing, such as mitochondrial DNA editing using mitoCas9.III. Nucleic acid constructs

[0120] A further aspect of the instant disclosure encompasses one or more expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed. The one or more expression constructs comprising a promoter operably linked to a nucleic acid sequence encoding a programmable nucleic acid modification system targeted to a target nucleic acid sequence. Expression of the nucleic acid modification system in the plant or part thereof, plant cell, or seed introduces a genetic modification in the target nucleic acid sequence. The geneticmodification confers an improved response to shade and higher temperatures to the Thlaspi sp. plant. In some aspects, the Thlaspi sp. plant is Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense. In some aspects, the pennycress plant is Thlaspi arvense (pennycress).

[0121] In some aspects, the one or more nucleic acid constructs encode the components of the engineered nucleic acid modification system described in Section II herein above.

[0122] Any of the multi-component systems described herein are to be considered modular, in that the different components can optionally be distributed among two or more nucleic acid constructs as described herein. The nucleic acid constructs can be DNA or RNA, linear or circular, single-stranded or double-stranded, or any combination thereof. The nucleic acid constructs can be codon-optimized for efficient translation into protein, and possibly for transcription into an RNA donor polynucleotide transcript in the cell of interest. Codon optimization programs are available as freeware or from commercial sources.

[0123] Expression constructs generally comprise DNA coding sequences operably linked to at least one promoter control sequence for expression in a cell of interest. Promoter control sequences can regulate the expression of various components, such as the programmable targeting nuclease in both bacterial (e.g., E. coli) and eukaryotic (e.g., yeast, insect, mammalian, or plant) cells. Suitable bacterial promoters include T7, lac operon, trp, and tac promoters, among others.

[0124] For eukaryotic systems, promoters can be constitutive, regulated, or tissue-specific. Constitutive promoters provide a range of expression levels, from weak to strong. Suitable eukaryotic constitutive promoter control sequences include, but are not limited to, the cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor (EDI )-alpha promoter, ubiquitin promoters, actin promoters, tubulin promoters, and immunoglobulin promoters. Plant-specific constitutive promoters include the CaMV35S promoter, CaMV 19S, GOS2, Arabidopsis At6669 promoter, Ricecyclophilin, Maize H3 histone, Synthetic Super MAS, opine promoters, plant ubiquitin (Ubi) promoters, actin 1 (Act-1 ) promoters, pEMU, Cestrum yellow leaf curling virus promoter (CYMLV promoter), and alcohol dehydrogenase 1 (Adh-1 ) promoter. Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026; 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0125] Non-constitutive promoters include tissue-preferred, tissue-specific, celltype specific, and inducible promoters. Examples of tissue-specific promoters are B29, CD14, CD43, CD45, CD68, desmin, elastase-1 , endoglin, fibronectin, Flt-1 , GFAP, GPIIb, ICAM-2, INF- , Mb, Nphsl, OG-2, SP-B, SYN1 , and WASP promoters. Plantspecific tissue-specific promoters include leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed-preferred promoters [e.g., from seed-specific genes (Simon et al., Plant Mol. Biol. 5. 191 , 1985; Scofield et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis et al., Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa et al., FEBS Letts. 221 : 43-47, 1987), Zein (Matzke et al., Plant Mol Biol, 143: 323-32, 1990), napA (Stalberg et al., Planta 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins et al., Plant Mol. Biol. 19: 873-876, 1992)], endosperm-specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b, and g gliadins (EMBO3: 1409-15, 1984), Barley ltd promoter, barley B1 , C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal, 116(1 ): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice-globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB / OHP-1 (Nakase et al., Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68,1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma-kafirin (PMB 32:1029-35, 1996)], embryo-specific promoters [e.g., rice OSH1 (Sato et al., Proc. Natl. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma et al., Plant Mol. Biol. 39:257-71 , 1999), rice oleosin (Wu et al., J. Biochem., 123:386, 1998)], and flowerspecific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245; 1989), apetala-3], TaGH9 from wheat Liqing Luo et al., (Int J Mol Sci. 2022 Jun; 23(11 ): 6324), truncated Ms2 promoter containing a TRIM element or a rice promoter OsLTP (Szabala Plant Cell Rep. 2023), the EC1 .1 promoter from Arabidopsis that drives the expression of genes specifically in the egg cell, and promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell (Koszegiet al., Plant J. 2011 ;67(2):280-91 ), the disclosures of all of which are incorporated herein by reference in their entirety.

[0126] Regulated promoters respond to various forms of environmental stresses or stimuli, including mechanical shock, heat, cold, flooding, drought, salt, anoxia, pathogens such as bacteria, fungi, and viruses, and nutritional deprivation. They can be induced by abiotic stresses like wounding, cold, desiccation, ultraviolet-B, heat shock, drought, or water stress, as well as biotic stresses like pathogen stress. Suitable regulated plant promoter sequences include salt-inducible promoters like RD29A, drought-inducible promoters like maize rab17, rab28, and Ivr2, and heat-inducible promoters like the tomato hsp80-promoter.

[0127] Promoters can also be plant-specific or used in plants, with a wide variety known to those skilled in the art. Any promoter sequence can be wild type or modified for more efficient expression. The DNA coding sequence can be linked to a polyadenylation signal (e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal) and / or a transcriptional termination sequence. In some cases, the complex or fusion protein can be purified from bacterial or eukaryotic cells.

[0128] The nucleic acid constructs can be used to express one or more components of the system for later introduction into a cell to be genetically modified. Alternatively, the nucleic acid constructs can be introduced into the cell to be geneticallymodified for expression of the components of the system in the cell. In some aspects, the nucleic acid constructs transiently express the various components of the system. Transiently expressing the system in a plant overcomes the cumbersome regulatory hurdles required for traditionally genetically modified crops. In some aspects, the engineered nucleic acid modification system is expressed in male reproductive tissues, modifies expression of various factors described herein above in male reproductive tissues, or both.

[0129] Any of the promoter sequences can be wild type or can be modified for more efficient or efficacious expression. The DNA coding sequence also can be linked to a polyadenylation signal (e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal, etc.) and / or at least one transcriptional termination sequence. In some situations, the complex or fusion protein can be purified from the bacterial or eukaryotic cells.

[0130] Nucleic acids encoding one or more components of an engineered DNA methylation system and / or transcription activation system can be present in a construct. Suitable constructs include plasmid constructs, viral constructs, and self-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254). For instance, the nucleic acid encoding one or more components of an engineered DNA methylation system and / or transcription activation system can be present in a plasmid construct.

[0131] Non-limiting examples of suitable plasmid constructs include plIC, pBR322, pET, pBluescript, and variants thereof. Alternatively, the nucleic acid encoding one or more components of an engineered DNA methylation system and / or transcription activation system can be part of a viral vector (e.g., lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, and so forth).

[0132] The plasmid or viral vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable reporter sequences (e.g., antibiotic resistance genes), origins of replication, T-DNA border sequences, and the like. The plasmid or viral vector can further comprise RNA processing elements such as glycine tRNAs, or Csy4 recognition sites. Such RNA processing elements can, forinstance, intersperse polynucleotide sequences encoding multiple gRNAs under the control of a single promoter to produce the multiple gRNAs from a transcript encoding the multiple gRNAs. When a cys4 recognition cite is used, a vector can further comprise sequences for expression of Csy4 RNAse to process the gRNA transcript. Additional information about vectors and use thereof can be found in “Current Protocols in Molecular Biology”, Ausubel et al., John Wiley & Sons, New York, 2003, or “Molecular Cloning: A Laboratory Manual”, Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0133] The plasmid or viral vector can also comprise a transit peptide for targeting of a protein product, particularly to a chloroplast, leucoplast or other plastid organelle or vacuole or an extracellular location. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, herein incorporated by reference in their entirety. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of other such isolated chloroplast proteins include, but are not limited to those associated with the small subunit (SSU) of ribulose- 1 ,5, -bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the lightharvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS) and transit peptides described in U.S. Pat. No.7,193,133, herein incorporated by reference. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP (CTP2, Klee et al., Mol. Gen. Genet. 210:437-442), and the Petunia hybrida EPSPS CTP (CTP4, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877) has been show to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants. The production of glyphosate tolerant plants by expression of a fusion protein comprising an amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well known by those skilled in the art, (U.S. Pat. No.5,627,061 , U.S. Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571 , EP 189707, EP 508909, and EP 924299).

[0134] In some aspects, the programmable nucleic acid modification system comprises a CRISPR / Cas nuclease system. The CRISPR / Cas nuclease system can be a CRISPR / Cas9 system comprising a CAS9 protein (CAS9) and a guide RNA (gRNA). A CRISPR / Cas9 system of the instant disclosure can be as described in Section II herein above. Accordingly, in some aspects, the one or more expression constructs of the instant disclosure comprise one or more expression constructs for expressing the CRISPR / Cas9 system. In some aspects, the one or more expression constructs for expressing the CRISPR / Cas9 system comprise an expression construct for expressing Cas9 nuclease, and an expression construct for expressing the guide RNA.

[0135] In some aspects, an expression construct for expressing Cas9 nuclease encodes a Cas9 nuclease encoded by a nucleic acid sequence comprising about 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% sequence identity with the nucleic acid sequence of SEQ ID NO: 8. In some aspects, an expression construct for expressing Cas9 nuclease encodes a Cas9 nuclease encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 8.

[0136] In some aspects, an expression construct for expressing Cas9 nuclease comprises a nucleic acid sequence encoding the Cas9 nuclease operably linked to a EC1 .1 promoter and an EC1 .2 enhancer. In some aspects, an expression construct comprising a nucleic acid sequence encoding the Cas9 nuclease operably linked to a Cre1 .1 promoter comprises a nucleic acid sequence comprising about 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% sequence identity with the nucleic acid sequence of SEQ ID NO: 9. In some aspects, an expression construct comprising a nucleic acid sequence encoding the Cas9 nuclease operably linked to a EC1.1 promoter comprises a nucleic acid sequence comprising at least about 75% or more, atleast about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 9.

[0137] In some aspects, an expression construct for expressing a guide RNA expresses a gRNA comprising a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof. In some aspects, an expression construct for expressing a gRNA comprises a nucleic acid sequence comprising about 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% sequence identity with the nucleic acid sequence of SEQ ID NO: 10. In some aspects, an expression construct for expressing a gRNA comprises a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 10.IV. Methods

[0138] One aspect of the instant disclosure encompasses a method of generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures. The method comprises introducing one or more expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed. The one or more expression constructs can be as described in Section III herein above. The one or more constructs comprise a promoter operably linked to a nucleic acid sequence encoding a programmable nucleic acid modification system targeted to a target nucleic acid sequence. Expression of the nucleic acid modification system in the plant or part thereof, plant cell, or seed introduces a genetic modification in the target nucleic acid sequence and wherein the genetic modification confers an improved response to shade and higher temperatures to the Thlaspi sp. plant. An engineered nucleic acid modification system can be as described in Section II herein above.

[0139] The method further comprises growing the Thlaspi sp. plant or part thereof, plant cell, or seed for a time and under conditions sufficient for the one or morenucleic acid expression constructs to express the programmable nucleic acid modification system in the Thlaspi sp. plant or part thereof, plant cell, or seed. Expressing the programmable nucleic acid modification system introduces a nucleic acid modification in the nucleic acid sequence encoding a PIF7 protein, thereby modifying the expression of the PIF7 protein, thereby generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising improved response to shade and higher temperatures to the Thlaspi sp. plant.

[0140] An additional aspect of the instant disclosure encompasses a method of improving response of a Thlaspi sp. plant or part thereof, plant cell, or seed to shade and higher temperatures of the Thlaspi sp. plant. The method comprises cultivating a genetically modified Thlaspi sp. plant, or a part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures, thereby improving performance of the Thlaspi sp. plant in culture. Improving performance of a Thlaspi sp. plant comprises improving the yield of a Thlaspi sp. plant in an agricultural system comprising sequential or overlapping cropping cycles, enhancing weed suppression, increased biomass, or any of the combinations thereof.

[0141] Another aspect of the instant disclosure encompasses a method of improving performance of a Thlaspi sp. plant in culture. The method comprises cultivating a genetically modified Thlaspi sp. plant, or a part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures, thereby improving performance of the Thlaspi sp. plant in culture. The genetically modified Thlaspi sp. plant, or a part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures can be as described in Section I herein above. Improving performance of a Thlaspi sp. plant can comprise improving the yield of a Thlaspi sp. plant in an agricultural system comprising sequential or overlapping cropping cycles, enhancing weed suppression, increased biomass, or any of the combinations thereof.

[0142]

[0143] Non-limiting examples of methods of introducing genetic modifications in a plant cell can be transposon insertion mutagenesis, T-DNA insertion mutagenesis, T-DNA activation tagging, chemically or radio-induced mutagenesis, TILLING (Targeted Induced Local Lesions In Genomes), site-directed mutagenesis, directed evolution, homologous recombination, introducing an engineered nucleic acid modification system such as a CRISPR / Cas system, or any combination thereof.

[0144] Methods for TILLING are well known in the art and include McCallum et al. (2000) Nat. Biotechnol. 18: 455-457; reviewed by Stemple (2004) Nat. Rev. Genet. 5(2): 145-50, the disclosures of all of which are incorporated herein in their entirety. In short, TILLING is a mutagenesis technology useful to generate and / or identify, and to eventually isolate, mutagenized plants. TILLING also allows selection of plants carrying such mutant plants. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis; (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product.

[0145] Populations or libraries of plants comprising genetic modifications can also be used in a method of the instant disclosure. When populations of plants comprising genetic modifications are used, the method can comprise the identification of a plant in the population comprising a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed. Non-limiting examples of populations of plants comprising genetic modifications include TILLING populations, SNP populations, populations of plants comprising naturally-occurring variations, or any combination thereof. Methods of screening populations of populations of plants comprising genetic modifications to identify are known in the art.

[0146] In some aspects, methods of introducing a nucleic acid modification of the instant disclosure comprise using an engineered nucleic acid modification system to generate the genetically modified plant. The methods can comprise introducing an engineered nucleic acid modification system or introducing nucleic acid constructsencoding the components of the engineered nucleic acid modification system. Engineered nucleic acid modification systems can be as described in Section II herein above, and nucleic acid constructs encoding components of the engineered nucleic acid modification systems can be as described in Section III herein above.

[0147] The engineered nucleic acid modification system modifies the expression of a nucleic acid sequence encoding a PIF7 protein in the plant. The plant or plant cell is then grown under conditions whereby the nucleic acid expression construct expresses the programmable nucleic acid modification system in the plant or plant cell. Expressing the programmable nucleic acid modification system or expressing the polypeptide or polynucleotide introduces a nucleic acid modification of the nucleic acid sequence encoding the polypeptide or polynucleotide, thereby modifying the expression of the polypeptide or polynucleotide in the plant. In some aspects, the engineered nucleic acid modification system is expressed in male reproductive tissues, modifies expression of various factors described herein above in male reproductive tissues, or both.(a) Introduction into the cell

[0148] The method comprises introducing a nucleic acid construct expressing an engineered protein into a cell of interest. As explained above, an engineered protein can be encoded on more than one nucleic acid sequence. Accordingly, a method of the instant disclosure comprises introducing more than one nucleic acid construct into the cell.

[0149] The one or more nucleic acid constructs described above can be introduced into the cell by a variety of means. Suitable delivery means include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposomes and other lipids, dendrimer transfection, heat shock transfection, nucleofection transfection, gene gun delivery, dip transformation, supercharged proteins, cell-penetrating peptides, viral vectors, magnetofection, lipofection, impalefection, optical transfection, Agrobacterium tumefaciens mediated foreign gene transformation, proprietary agent-enhanced uptakeof nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. The choice of means of introducing the system into a cell can and will vary depending on the cell, or the system or nucleic acid nucleic acid constructs encoding the system, among other variables.(b) Culturing a cell

[0150] The method further comprises culturing a cell under conditions suitable for expressing the engineered protein. Methods of culturing cells are known in the art. In some aspects, the cell is from an animal, fungi, oomycete or prokaryote. In some aspects, the cell is a plant cell, plant, or plant part. When the cell is in tissue ex vivo, or in vivo within a plant or within a plant part, the plant part and / or plant can also be maintained under appropriate conditions for insertion of the donor polynucleotide. In general, the plant, plant part, or plant cell is maintained under conditions appropriate for cell growth and / or maintenance. Those of skill in the art appreciate that methods for culturing plant cells are known in the art and can and will vary depending on the cell type. Routine optimization can be used, in all cases, to determine the best techniques for a particular cell type. See for example, in Santiago et al. (2008) PNAS 105:5809- 5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646- 651 ; Lombardo et al. (2007) Nat. Biotechnology 25:1298-1306; and Taylor et al. (2012) Tropical Plant Biology 5:127-139.V. Kits

[0151] A further aspect of the present disclosure provides kits for generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures. The kits comprise one or more genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures; one or more expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; one or more plants or plant cells comprising one or more expression constructsfor introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; or any combination thereof. The genetically modified plant can be as described in Section I herein above, the engineered nucleic acid modification system can be as described in Section II herein above, the one or more nucleic acid constructs encoding the components of the engineered nucleic acid modification system can be as described in Section III herein above.

[0152] The kits can further comprise transfection reagents, cell growth media, selection media, in vitro transcription reagents, nucleic acid purification reagents, protein purification reagents, buffers, and the like. The kits provided herein generally include instructions for carrying out the methods detailed below. Instructions included in the kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.DEFINITIONS

[0153] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991 ); and Hale & Marham, The Harper Collins Dictionary of Biology (1991 ). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0154] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0155] A “genetically modified” plant refers to a plant in which the nuclear, organellar or extrachromosomal nucleic acid sequences of a cell has been modified, i.e. , the cell contains at least one nucleic acid sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide.

[0156] As used herein, the term "gene" refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0157] As used herein, the term “engineered” when applied to a targeting protein refers to targeting proteins modified to specifically recognize and bind to a nucleic acid sequence at or near a target nucleic acid locus. A “genetically modified” plant refers to a cell in which the nuclear, organellar or extrachromosomal nucleic acid sequences of a cell have been modified, i.e., the cell contains at least one nucleic acid sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide.

[0158] The term “nucleic acid modification” refers to processes by which a specific nucleic acid sequence in a polynucleotide is changed such that the nucleic acid sequence is modified. The nucleic acid sequence can be modified to comprise an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide. The modified nucleic acid sequence isinactivated such that no product is made. Alternatively, the nucleic acid sequence can be modified such that an altered product is made.

[0159] As used herein, the term "mutant" or "mutation" refers to a genetic alteration in a plant that results in a change from the genetic background of the parent plant from which the mutant was generated. This alteration can involve an insertion, deletion, or substitution of nucleotides within the plant's DNA, leading to a modification in gene expression or function. In the context of this disclosure, mutations are specifically introduced to modify the expression of the PIF7 gene in Thalspi sp. plants such as pennycress, resulting in plants with improved responses to environmental conditions such as shade and elevated temperatures. These mutations are generated from a wild-type genetic background, providing a basis for comparison to assess the phenotypic changes and advantages conferred by the genetic modification.

[0160] As used herein, “protein expression” includes but is not limited to one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); production of a mutant protein comprising a mutation that modifies the activity of the protein, including the calcium channel activity; and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0161] The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties. In general, an analog of a particular nucleotide has the same base-pairing specificity, i.e., an analog of A will base-pair with T. The nucleotides of a nucleic acid or polynucleotide can be linked by phosphodiester, phosphothioate, phosphoram idite, phosphorodiamidate bonds, or combinations thereof.

[0162] The term "nucleotide" refers to deoxyribonucleotides or ribonucleotides. The nucleotides can be standard nucleotides (i.e. , adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog can be a naturally occurring nucleotide (e.g., inosine) or a non- naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines). Nucleotide analogs also include dideoxy nucleotides, 2’-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[0163] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.

[0164] The terms “phenotype”, or “phenotypic trait” or “trait” refer to one or more traits of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait”. In other cases, a phenotype is the result of several genes.

[0165] A “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or progeny of the same. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.

[0166] As used herein, the terms "target site", "target sequence", or “nucleic acid locus” refer to a nucleic acid sequence that defines a portion of a nucleic acid sequence to be modified or edited and to which a homologous recombination composition is engineered to target.

[0167] As used herein, the terms "foliar shade" or "shade" refer to a specific light condition characterized by a reduction in the red:far red light ratio, which is a key factor influencing plant growth responses. In this context, foliar shade is defined as a level of shade where the red:far red ratio is approximately 0.1 , simulating the light environment beneath a canopy where the red light is absorbed by the leaves above, and the far-red light is more prevalent. This condition is significant as it triggers shade avoidance responses in plants, such as elongation, which are undesirable for agricultural productivity. The term encompasses light conditions that mimic the natural shading effects experienced by plants inter-seeded with taller crops, such as corn, during late fall planting.

[0168] As used herein, the term "elevated temperature" refers to temperature conditions that exceed the typical or optimal range for the growth and development of pennycress plants. Specifically, elevated temperatures are those that can induce stress responses in plants, such as increased elongation or other morphogenic changes that are not desirable for agricultural productivity. Cultivation temperatures that can be considered elevated for pennycress can range from about 25°C to about 30°C, from about 28°C to about 35°C, or even from about 30°C to about 40°C or higher. These temperatures mimic conditions that pennycress might encounter during late fall planting, where higher temperatures can exacerbate elongation responses and affect plant establishment and yield.

[0169] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between twoaligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482- 489 (1981 ). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. 0. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80- 82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.

[0170] As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.EXAMPLES

[0171] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0172] The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0173] The following examples are included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.Example 1. Pennycress is shade and heat intolerant

[0174] The response of pennycress to foliar shade and temperature was tested in seedlings. Seedlings were grown under white light conditions, foliar shade (similar photosynthetically active radiation, but a red:far red ratio of 0.1 ), at an elevated temperature of 28°C, and a combination of foliar shade and 28°C. The results show that pennycress is sensitive to foliar shade and heat (FIGs. 1A-1G).Example 2. Generation of Pif7 loss of function mutations in pennycress

[0175] Two loss of function mutations in the PIF7 gene in pennycress were generated using a CRISPR / Cas9 nucleic acid modification system. The PIF7 protein is a bHLH transcription factor, involved in regulating foliar shade avoidance responses.

[0176] Two guide RNAs specific to target PIF7 were designed to generate a deletion resulting from cellular repair of a targeted DNA break in the absence of an externally provided repair template. The two guide RNAs were designed to specifically target PIF7 and are predicted to have no off-target sites.

[0177] Plants were transformed with pHEE401 , a plasmid comprising constructs for expressing the PIF7 specific guide RNAs, the CRISPR / Cas system, and a hygromycin resistance gene. Transformants were initially identified by selection for resistance to hygromycin. The CRISPR / Cas 9 nuclease made a targeted double stranded break at both sites complementary to the guide RNAs in positive transgenics. The endogenous DNA repair mechanisms repaired the DNA breaks by removing the sequence between the initial breaks, resulting in a small deletion in the PIF7 gene. Two additional mutations were also generated. Sequencing of PIF7 confirmed the modifications. The three loss of function mutations are shown in FIG. 2.

[0178] A line with the absence of CRISPR / Cas and presence of the PIF7 edit was selected from a segregating population. Progeny from this line was screened for sensitivity to hygromycin. The final edited line is free from exogenous DNA. Tiling PCR of the entire transgene spanning the left and right border of the T-DNA and plasmid backbone was performed on pooled DNA from dozens of progeny to confirm absence of foreign DNA.Example 3. Effect of loss of function of PIF7 on pennycress phenotype

[0179] Evaluation of CRISPR and EMS alleles of PIF7 show that pif7 plants have reduced hypocotyl length (FIGs. 3A and 3B). To test the effect of foliar shade and temperature conditions on adult phenotypes of pennycress, plants were germinated and grown for 4 d under control conditions (22 °C 16 hrs light / 8 hrs dark), then grown for an additional 14 d under treatment conditions. The plants were then rescued to controlconditions until senescence. There were 18 plants for each treatment. Shade conditions included the addition of Far-red light 16 hrs during day and heat conditions were 28 °C for 16 hrs during day. Shade + heat conditions comprised Far red + 28 °C for 16 hrs during day. The traits that were measured included rosette morphology, flowering time, branching, and yield.

[0180] The pif7 null mutant plants show improved phenotypes when compared to wt plants (FIGs. 4A-4E). Mutant plants also showed a slight delay in flowering in response to stress (FIG. 5).

[0181] Yields of wt and mutant plants were measured. The results show that manipulating PIF7 does not decrease yield under white light and foliar shade conditions (FIG. 6)Example 4. Unique response of pennycress

[0182] The inventors discovered that pennycress has unique responses to combined foliar shade and heat stresses when compared to related plants such as Arabidopsis. Shade and elevated temperature together have a repressive effect on hypocotyl elongation compared to heat treatment alone (FIG. 3B). This is the opposite of what is reported in Arabidopsis, where foliar shade and elevated temperature are synergistic and hypocotyls elongate more in combined conditions than either foliar shade or temperature alone (Burko et al, Nat Commun. 2022 Aug 29; 13(1 ):4942).

[0183] Pennycress roots have comparable root length in white light and foliar shade conditions (FIGs 7A-7E). Other species have described reduced root length in foliar shade. Pif7 plants have increased total root length, primary root length, and lateral root length in foliar shade conditions which is more desirable as it indicates the plants are not partitioning resources to only above ground growth.Example 5. Effects of other light sensors in pennycress

[0184] Interestingly, ELF3 mutants, which alter phytochrome signaling and which has been found to affect response of related plants to light, do not decrease hypocotyl length (FIG. 8).Example 6. Loss of function pif7 mutants have reduced hypocotyl elongation across genetic backgrounds

[0185] Effects of pif7 loss of function mutations on response to lower light conditions and elevated temperatures were tested across a number of genetic backgrounds. The results show that loss of function p / 77 plants have reduced hypocotyl elongation across all genetic backgrounds tested (FIGs. 3B and 9A-9C).Example 7. Loss of function pif7 mutants have reduced organ elongation in lower light conditions

[0186] Effects of p / 77 loss of function mutations on organ elongation in response to lower light conditions were tested across a number of genetic backgrounds. The results show that loss of function p / 77 plants have reduced hypocotyl elongation and petiole length (FIGs. 10A-10E).Example 8. Chlorophyll fluorescence of plants grown in shade and heat.

[0187] Effects of p / 77 loss of function mutations on chlorophyll inflorescence in response to lower light conditions and elevated temperatures were tested across a number of genetic backgrounds. The results show that loss of function p / 77 plants have a comparable Fv / Fm to wild type plants indicating that they are still healthy. NPQ, NDVI and CIRE was either improved when compared to wild type or statistically similar (FIGs.11A-11D).Example 9. pif7 maintains ecosystem services without a drag on yield

[0188] Effects of pif7 loss of function mutations on canopy closure, solidity, convex hull area, inflorescence, and yield were measured in mini fields. The results show that loss of function pif7 plants have reduced elongation when planted at high density while maintaining flowering time and yield (FIGs. 12A-12F).SEQUENCES

Claims

CLAIMSWhat is claimed is:1 . A genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response to shade and higher temperatures, the genetically modified plant comprising a genetic modification of at least one target nucleic acid sequence, wherein the genetic modification modifies expression of a phytochrome-interacting factor 7 (PIF7) protein, wherein the genetic modification confers an improved response to shade and higher temperatures to the Thlaspi sp. plant.

2. The genetically modified Thlaspi sp. plant of any one of the preceding claims, wherein the PIF7 protein is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

3. The genetically modified Thlaspi sp. plant of claim 1 or claim 2, wherein the genetic modification is in a nucleic acid sequence encoding the PIF7 protein.

4. The genetically modified Thlaspi sp. plant of any one of the preceding claims, wherein the genetic modification is in an endogenous nucleic acid sequence encoding the PIF7 protein.

5. The genetically modified Thlaspi sp. plant of any one of the preceding claims, wherein the genetic modification is a loss of function mutation.

6. The genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed of any one of the preceding claims, wherein the Thalspi sp. plant is Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense.

7. The genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed of any one of the preceding claims, wherein the Thalspi sp. plant is Thlaspi arvense.

8. The genetically modified Thlaspi sp. plant of claim 7, wherein the genetic modification that modifies expression of the PIF7 protein is a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein.

9. The genetically modified Thlaspi sp. plant of claim 8, wherein the endogenous nucleic acid sequence comprising the loss of function mutation comprises at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or any combination thereof.

10. The genetically modified Thlaspi sp. plant of any one of the preceding claims, wherein the modification of the polynucleotide encoding the PIF7 protein comprises a modification introduced by a programmable nucleic acid modification system comprising a CRISPR / Cas nuclease system, wherein the CRISPR / Cas nuclease system comprises a CAS9 protein (CAS9) and a guide RNA (gRNA).11 . The genetically modified Thlaspi sp. plant of claim 7, wherein the Thlaspi sp. is Thlaspi arvense and the gRNA comprises a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof.

12. The genetically modified Thlaspi sp. plant of any one of the preceding claims, wherein the genetically modified plant is a Thlaspi arvense plant comprising a loss of function mutation in an endogenous nucleic acid sequence encoding the PIF7 protein and wherein the endogenous nucleic acid sequence encoding the PIF7 protein comprising the loss of function mutation comprises at least about 75% or more, at least about 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% sequence identity with a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

13. One or more expression constructs for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp.plant or part thereof, plant cell, or seed, the one or more expression constructs comprising a promoter operably linked to a nucleic acid sequence encoding a programmable nucleic acid modification system targeted to a target nucleic acid sequence, wherein expression of the nucleic acid modification system in the plant or part thereof, plant cell, or seed introduces a genetic modification in the target nucleic acid sequence and wherein the genetic modification confers an improved response to shade and higher temperatures to the Thlaspi sp. plant.

14. The one or more expression constructs of claim 13, wherein the Thlaspi sp. plant is Thlaspi perfoliatum, Thlaspi caerulescens, or Thlaspi arvense.

15. The one or more expression constructs of claim 13 or claim 14, wherein the pennycress plant is Thlaspi arvense (pennycress).

16. The one or more expression constructs of any one of claims 13-15, wherein the programmable nucleic acid modification system comprises a CRISPR / Cas nuclease system.

17. The one or more expression constructs of claim 16, wherein the CRISPR / Cas nuclease system comprises a CAS9 protein (CAS9) and a guide RNA (gRNA).

18. The one or more expression constructs of claim 17, wherein the Cas9 nuclease is encoded by a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with an amino acid sequence of SEQ ID NO: 8.

19. The one or more expression construct of claim 17 or claim 18, wherein the Thlaspi sp. is Thlaspi arvense and the gRNA comprises a nucleic acid sequence of SEQ ID NO: 11 , SEQ ID NO: 12, or a combination thereof.

20. The one or more expression constructs of any one of claims 17-19, wherein the one or more expression constructs for expressing the CRISPR / Cas9 system comprise an expression construct for expressing Cas9 nuclease, and an expression construct for expressing the guide RNA.21 . The one or more expression constructs of any one of claims 17-20, wherein the expression construct for expressing the Cas9 nuclease comprises a nuclei acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 9.

22. The one or more expression constructs of any one of claims 17-21 , wherein the expression construct for expressing the gRNA comprises a nucleic acid sequence comprising at least about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 10.

23. A Thlaspi sp. plant or plant cells comprising one or more expression constructs of claims 13-22.

24. A method of generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures, the method comprising: a. introducing one or more expression constructs of any one of claims 13-22 into a Thlaspi sp. plant or part thereof, plant cell, or seed; and b. growing the Thlaspi sp. plant or part thereof, plant cell, or seed for a time and under conditions sufficient for the one or more nucleic acid expression constructs to express the programmable nucleic acid modification system in the Thlaspi sp. plant or part thereof, plant cell, or seed; wherein expressing the programmable nucleic acid modification system introduces a nucleic acid modification in the nucleic acid sequence encoding a PIF7 protein, thereby modifying the expression of the PIF7 protein, thereby generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising improved response to shade and higher temperatures to the Thlaspi sp. plant.

25. A method of improving response of a Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures, the method comprising generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed exhibiting an improved response to shade and higher temperatures of any one of claims 1 -12 using a method of claim 24.

26. A method of improving performance of a Thlaspi sp. plant in culture, the method comprising cultivating a genetically modified Thlaspi sp. plant, or a part thereof, plant cell, or seed exhibiting an improved response of the Thlaspi sp. plant to shade and higher temperatures of any one of claims 1 -12, thereby improving performance of the Thlaspi sp. plant in culture, wherein improving performance of a Thlaspi sp. plant comprises improving the yield of a Thlaspi sp. plant in an agricultural system comprising sequential or overlapping cropping cycles, enhancing weed suppression, increased biomass, or any of the combinations thereof.

27. A kit for generating a genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures, the kit comprising: a. one or more genetically modified Thlaspi sp. plant or part thereof, plant cell, or seed comprising an improved response to shade and higher temperatures of claims 1 -12; b. one or more expression constructs of any one of claims 13-22 for introducing a genetic modification that confers an improved response to shade and elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; c. Thlaspi sp. plant or plant cells of claim 23 comprising expression constructs for introducing a genetic modification that confers an improved response to shadeand elevated temperatures to a Thlaspi sp. plant or part thereof, plant cell, or seed; or d. any combination of (a)-(c).