Method for screening plant function gain mutation and composition thereof

Abstract

The present disclosure relates to methods of screening for functional gain mutations in a non-coding region of a target gene. The target gene may be an NPQ gene that includes a photosystem II subunit S (PsbS), a zeaxanthin epoxidase (ZEP), and a violaxanthin deepoxidase (VDE). The disclosure further relates to a method of improving an economic crop plant or crop seed by introducing a functional gain mutation in a non-coding region of a target gene, and an improved economic crop plant or crop seed produced by the method.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 329,831, filed April 11, 2022, which is incorporated herein by reference in its entirety. References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing (794542001940SEQLIST.xml; size: 171,268 bytes; and creation date: April 11, 2023) are hereby incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to a method for screening for a gain-of-function mutation in a non-coding region of a target gene. The target gene may be a NPQ gene, which includes photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP) and violaxanthin de-epoxidase (VDE). The present application further relates to a method for improving cash crop plants or crop seeds by introducing a gain-of-function mutation in a non-coding region of a target gene, and an improved cash crop plant or crop seed produced by the method. Background Art

[0004] Optimizing photosynthetic efficiency remains one of the largest unfulfilled gaps in engineering sustainable and high-yield crop varieties (Long SP, Marshall-Colon A, Zhu XG. Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell. 2015; 161(1): 56-66. doi: 10.1016 / j.cell.2015.03.019). Previous studies have shown that overexpression of genes involved in non-photochemical quenching (NPQ), a photoprotective process necessary to dissipate excess absorbed light energy, can improve photosynthetic efficiency, biomass accumulation (Kromdijk J, Glowacka K, Leonelli L et al., Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science. 2016; 354(6314): 857-862) and water use efficiency (Glowacka K, Kromdijk J, Kucera K et al., Photosystem II Subunit Soverexpression increases the efficiency of water use in a field-growing crop. Nat Commun. 2018; 9(868): 1-9. doi: 10.1038 / s41467-018-03231-x) in the model crop Nicotiana tabacum under field-relevant conditions. In addition, it has been shown that overexpression of genes involved in NPQ increases soybean seed yield (Souza APD, Burgess SJ, Doran L., Hansen J., Manukyan L., Maryn N. et al., (2022). Soybean photosynthesis and crop yield are improved by accelerating recovery from photoprotection. Science 377, 851-854. doi: 10.1126 / science.adc9831).However, these approaches rely on the expression of exogenous DNA or transgenes, which introduce additional regulatory complexity and may be susceptible to transgenerational gene silencing (James VA, Avart C, Worland B, Snape JW, Vain P. The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet. 2002; 104(4): 553-561. doi: 10.1007 / s001220100745).

[0005] CRISPR / Cas9 has greatly expanded the ability to generate targeted loss-of-function mutations. Recently, CRISPR / Cas9 editing of cis-regulatory elements has also been demonstrated by using this approach in tomato (Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett ME, Lippman ZB. Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing. Cell. 2017; 171(2): 470-480.e8.doi: 10.1016 / j.cell.2017.08.030) and maize (Liu L, Gallagher J, Arevalo ED et al., Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes.NatPlants.2021;7(3):287-294.doi:10.1038 / s41477-021-00858-5) produces partial loss-of-function alleles to reduce rather than eliminate the usefulness of gene expression. However, the ability to increase gene expression without using persistent transgenes is still lacking, and more sophisticated genome engineering through lead editing is limited by bottlenecks of low editing, transformation and regeneration efficiency (Wada N, Ueta R, Osakabe Y, Osakabe K. Precision genome editing in plants: state-of-the-art in CRISPR / Cas9-based genome engineering.2020:1-12). A powerful channel to achieve such gains will rapidly accelerate the possibility of gene editing in crop plants such as rice.

[0006] Methods for screening plants (which can be screened for phenotypes such as increased yield, quality, or both) for alleles with "weak effects" are described in US patent application US2020 / 0199604. The methods therein rely on the use of plants with a first allele (which is a hypomorphic allele or null allele) to allow screening for "weak effects", which limits the usefulness of the screening method.

[0007] NPQ genes represent a promising approach for genome editing methods across plant species, as it is well known that NPQ genes are found in all plants and NPQ proteins are highly conserved in their function. There is a need for genetic engineering methods that alter endogenous gene expression in order to achieve agronomic gains without the need for stable transgenes. In particular, there is a need for such methods that can enhance photoprotection (e.g., by targeting specific NPQ components) in order to improve photosynthetic processes and ultimately plant yield. Overview

[0008] To meet these needs, the present disclosure provides methods for screening for gain-of-function mutations in target genes, such as NPQ genes (e.g., PsbS, VDE, ZEP) to produce plants with improved photosynthetic processes. For example, the present disclosure uses this method to target the rice photosystem II subunit S (OsPsbS1) gene, a core factor for high light and fluctuating light tolerance, to produce mutations with increased OsPsbS1 expression, improved NPQ capacity, and presumably increased water use efficiency.

[0009] Aspects of the present disclosure include methods for screening a target gene of a plant for a gain-of-function mutation, the method comprising: (a) generating a set of mutations in a non-coding sequence (NCS) of a target gene in a plant cell population of a plant using one or more RNA-guided nucleic acid modification enzymes targeting the target gene, the one or more RNA-guided nucleic acid modification enzymes comprising one or more different guide RNAs; (b) regenerating the plant cell population into two or more plants that are hemizygous for the generated mutations; (c) (1) selfing the two or more plants to produce progeny plants, and (2) optionally, screening progeny plants that are homozygous for the mutations for screening in part (d); and (d) screening the progeny plants from step (c) to identify gain-of-function mutations. Additional embodiments of this aspect further include: (e) selecting plants with gain-of-function mutations, and (f) sequencing the target gene to identify gain-of-function mutations. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the one or more RNA-guided nucleic acid modification enzymes are expressed by an expression vector comprising a selectable marker, and the screening in step (d) includes screening plants that lack the selectable marker. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the gain-of-function mutation induces overexpression of the target gene. In a further embodiment of this aspect, the overexpression of the target gene is in the morning. In another embodiment of this aspect, the overexpression of the target gene is not constitutive. In still another embodiment of this aspect, the plant has a constitutive phenotype. In yet another embodiment of this aspect (which may be combined with any of the preceding embodiments), the target gene induction is related to photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield and CO2 One or more relevant phenotypes in the fixation, and the screening of step (c)(2) includes screening the progeny plants by chlorophyll fluorescence to identify the non-transgenic plants that are assumed to be homozygous for the mutation. In still another embodiment of this aspect (which can be combined with any of the preceding embodiments), the target gene induces a phenotype related to water use efficiency, and the screening of step (c)(2) includes screening the progeny plants by chlorophyll fluorescence to identify the non-transgenic plants that are assumed to be homozygous for the mutation. In still another embodiment of this aspect (which can be combined with any of the preceding embodiments), the method does not include the use of plants with hypomorphic alleles or null alleles of the target gene. In further embodiments of this aspect (which can be combined with any of the preceding embodiments), the gain-of-function mutation improves the yield, quality, or both of the plant with the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions. In additional embodiments of this aspect (which may be combined with any of the preceding embodiments), the one or more RNA-guided nucleic acid modification enzymes targeted to the target gene include two or more different guide RNAs, three or more different guide RNAs, four or more different guide RNAs, five or more different guide RNAs, ten or more different guide RNAs, or twenty or more different guide RNAs.

[0010] In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), each guide RNA targets a region of a target gene selected from the group consisting of a promoter region, an upstream regulatory region, a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), an intron, a micro-RNA binding site, an alternative splicing element, and a downstream regulatory element. In a further embodiment of this aspect, the guide RNA targets the 5'UTR of the target gene. In another embodiment of this aspect (which can be combined with any of the preceding embodiments), the guide RNA targets at least one region in the target gene that is at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical across plant species. In another embodiment of this aspect (which can be combined with any of the preceding embodiments), at least 50% of the mutation group is located in a region of the target gene selected from the group consisting of a promoter region, an upstream regulatory region, a 5'UTR, a 3'UTR, an intron, a micro-RNA binding site, an alternative splicing element, and a downstream regulatory element. In a further embodiment of this aspect, at least 50% of the mutation group is located in the 5'UTR of the target gene. In still another embodiment of this aspect (which can be combined with any of the preceding embodiments), the gain-of-function mutation is a deletion, an inversion, a translocation, an insertion, a transition, a transversion, or a combination thereof. In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), the gain-of-function mutation is, in each case, an increase in transcription of the target gene, an increase in the stability of the mRNA produced by the target gene, an increase in the translation of the protein coding region of the mRNA, or a decrease in mRNA degradation compared to a plant lacking the gain-of-function mutation grown under the same conditions. In another embodiment of this aspect (which can be combined with any of the preceding embodiments), one or more RNA-guided nucleic acid modification enzymes are Cas enzymes, base editors, or lead editors. In a further embodiment of this aspect, the Cas enzyme is selected from the group consisting of Cas9, Cas12, Cas12a, Cas13, Cas14, CasX, or CasY.

[0011] In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with Crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a maize plant, a biomass plant, an Arabidopsis thaliana plant, a tobacco (Nicotiana tabacum) plant, a rice (Oryza sativa) plant, a corn (Zea mays) plant, a sorghum (Sorghum bicolor) (sweet sorghum or grain sorghum) plant, a soybean (Glycine max) plant, a cowpea (Vigna unguiculata) plant, a poplar (Populus spp.) plant, a eucalyptus (Eucalyptus spp.) plant, a cassava (Manihot esculenta) plant, a barley (Hordeum vulgare plants, potato (Solanum tuberosum) plants, sugarcane (Saccharum spp.) plants, alfalfa (Medicago sativa) plants, Miscanthus plants, energy sugarcane plants, elephant grass plants, wheat plants, oat plants, oil palm plants, safflower plants, sesame plants, flax plants, cotton plants, sunflower plants, camelina plants, rapeseed (Brassica napus) plants, Ethiopian mustard (Brassica carinata) plants, mustard (Brassica juncea) plants, pearl millet plants, millet plants, other cereal plants, oilseed plants, vegetable crop plants, forage crop plants, industrial crop plants or woody crop plants. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the plant is an elite line or strain.

[0012] In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the target gene is selected from the group consisting of a photosystem II subunit S (PsbS) gene, a zeaxanthin epoxidase (ZEP) gene, and a violaxanthin de-epoxidase (VDE) gene. In additional embodiments of this aspect, the screening comprises evaluating one or more of the following: photosynthetic efficiency under fluctuating light conditions; photoprotection efficiency under fluctuating light conditions; increase in non-photochemical quenching (NPQ) induction rate under fluctuating light conditions; increase in non-photochemical quenching (NPQ) relaxation rate under fluctuating light conditions; improvement in quantum yield under fluctuating light conditions, and CO under fluctuating light conditions. 2Improved fixation. In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a PsbS gene, and wherein the PsbS gene comprises a sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:97 or SEQ ID NO:98. In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a ZEP gene, and the ZEP gene comprises SEQ ID NO:92. In further embodiments of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a VDE gene, and wherein the VDE gene comprises a sequence selected from the group consisting of SEQ ID NO:92 or SEQ ID NO:95. In yet another embodiment of this aspect, the one or more different guide RNAs include a spacer sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.In still another embodiment of this aspect, the one or more different guide RNAs comprise a spacer sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In a further embodiment of this aspect, the one or more different guide RNAs comprise a spacer sequence selected from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65. In yet another embodiment of this aspect (which can be combined with any of the aforementioned embodiments with an intervening sequence), one or more different guide RNAs are introduced using a vector, and wherein the vector comprises two or more gRNA scaffolds comprising SEQ ID NO: 9, and two or more tRNA linkers comprising SEQ ID NO: 10.

[0013] Some aspects of the present disclosure relate to methods for producing improved cash crop plants or crop seeds, the methods comprising: (a) selecting cash crop plants for improvement; (b) introducing a gain-of-function mutation identified in any of the methods of the foregoing embodiments into at least one cell of a cash crop to produce an improved cash crop cell; and (c) producing improved cash crop plants or crop seeds from improved cash crop plant cells. In further embodiments of this aspect, the cash crop plants include rice (Oryza sativa) plants, corn (Zea mays) plants, or cowpea (Vigna unguiculata) plants.

[0014] Additional aspects of the present disclosure include improved economic crop plants or crop seeds comprising a gain-of-function mutation in a non-coding sequence of a target gene, wherein the target gene induces photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO 2The invention relates to a phenotype related to one or more of fixation and water use efficiency, and the gain-of-function mutation improves the yield, quality, or both of the plant having the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions. Further embodiments of this aspect include cash crop plants including rice (Oryza sativa) plants, corn (Zea mays) plants, or cowpea (Vigna unguiculata) plants. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the target gene is selected from the group consisting of a photosystem II subunit S (PsbS) gene, a zeaxanthin epoxidase (ZEP) gene, and a violaxanthin de-epoxidase (VDE) gene.

[0015] Further aspects of the present disclosure include modified plants comprising an inversion in a cis-regulatory element of a PsbS gene, wherein the inversion increases PsbS gene expression. In a further embodiment of this aspect, the plant is a rice (Oryza sativa) plant. In still another embodiment of this aspect, the rice plant is a japonica rice subspecies (Oryza sativa ssp. Japonica) plant. In yet another embodiment of this aspect (which may be combined with any of the preceding embodiments), the increased PsbS gene expression compared to a control plant without an inversion in a cis-regulatory element of the PsbS gene includes overexpression, increased expression at one or more specific times, increased expression in one or more specific tissues, increased expression at one or more developmental stages, or a combination thereof. In still another embodiment of this aspect (which may be combined with any of the preceding embodiments), the inversion encompasses a portion of the 5'UTR of the PsbS gene. In another embodiment of this aspect, which may be combined with any of the preceding embodiments, the inversion comprises between 1,000 and 500,000 nucleotides, between 2,000 and 400,000 nucleotides, between 3,000 and 300,000 nucleotides, between 1,000 and 10,000 nucleotides, between 2,000 and 8,000 nucleotides, between 3,000 and 5,000 nucleotides, or about 4,000 nucleotides. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant or its ancestors is screened for inversions in the cis-regulatory elements of the PsbS gene and increased expression of the PsbS gene. In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the inversions in the cis-regulatory elements of the PsbS gene are randomly generated in the plant or its ancestors. In still another embodiment of this aspect, the inversions are randomly generated using guide RNAs.In a further embodiment of this aspect, the guide RNA comprises a spacer sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased PsbS gene expression results in photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO. 2 In another embodiment of this aspect, the increased PsbS gene expression comprises overexpression of PsbS in the morning, or is not constitutive overexpression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] This patent or application file contains at least one drawing printed in color. Copies of this patent or application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Figure 1A-1BTarget guide RNA (gRNA) sites for CRISPR / Cas9 mutagenesis upstream of OsPsbS1 in Oryza sativa subspecies (ssp.) japonica are shown. Figure 1A Target gRNA sites (triangles) distributed in the upstream distal (dark grey) and proximal (grey) regions of OsPsbS1 in Oryza sativa ssp. indica (top) and Oryza sativa ssp. japonica (bottom) and their positions relative to the OsPsbS1 cis-regulatory element (CRE) comprising the upstream promoter (black) and 5' untranslated region (5' UTR, white). The internal 2.7 kb O. sativa ssp. japonica-specific insertion is shown in grey. Figure 1B The number of lines recovered (on the x-axis) for each event (on the y-axis) of twenty-three independent transformants is shown.

[0018] Figures 2A-2F High-throughput screening and editing of NPQ in T 1 Transgenic segregation of progeny. Figure 2A Showing Mendelian segregation of cis-regulated OsPsbS1 editing (purple) and inheritance of the hemizygous Cas9 transgene. 0 In the group (top), plants contain a mutated cis-regulatory element (gray) and a transgene carrying Cas9 (represented by a gray shape; Cas9 icon from BioRender). 1 In the group (bottom), 25% of the plants are expected to be homozygous for each mutated cis-regulatory element (indicated by "A / A" (light gray square on upper left) and "a / a" (light gray square on lower right)), and 25% of the plants are expected to have lost the transgene carrying Cas9 (indicated by a gray prohibition sign). Figure 2B Shown are the PsbS knockout (0 on the x-axis), heterozygous (1 on the x-axis), and WT (2 on the x-axis) lines (n = 95, 117, and 101 biological replicates, respectively). - 2 s -1 Linear regression of the maximum NPQ capacity (on the y-axis) after 10 min of blue light exposure. Figure 2C Display T 0 Maximum NPQ (on the x-axis) and the corresponding T 1 The mean maximum NPQ of the alleles (on the y-axis) was weakly correlated (n = 78 T 0 Plants, each T 1 The groups consisted of 2-4 biological replicates. 0Generation, T 1 Individuals with NPQs that were +2SD above WT in one or both generations are shown in the lower right quadrant, upper left quadrant, and upper right quadrant, respectively. Figure 2D Shown is the chlorophyll fluorescence phenotyping of NPQ (on the y-axis) over time (on the x-axis; 10 min light followed by 10 min dark) using leaf punches to resolve single isolate T relative to WT (n=4, diamonds). 0 Homozygous alleles in 36 progeny of a parent (A / A, inverted triangles; a / a, squares). Heterozygous alleles were also resolved (A / a, triangles). Data shown are mean ± SEM. Figure 2E showed that adding the plant-selective antibiotic hygromycin to Figure 2D In leaf punches analyzed in the middle phenotyping, F cells lacking the Cas9 transgene were identified as having suppressed v / F m Sensitive individuals are distinguishable from resistant individuals that include the Cas9 transgene (grey) and are similar to wild type (grey). Figure 2F Representative images of chlorophyll fluorescence phenotyping are shown. The upper panel shows leaf perforations of WT (upper row) and T before antibiotic treatment. 1 The lower panel shows the leaf perforation of WT (upper panel) and T after 72 h of antibiotic treatment. 1 The leaves of the series are perforated (bottom three rows). The shading corresponds to the F v / F m (au), lighter shading indicates lower.

[0019] Figure 3A-3B Display spans 78 T 0 Maximum NPQ of the 120 putative homozygous alleles of the event. Figure 3A It shows that at 1500 μmol m -2 s -1 Maximum NPQ (on the y-axis) of 120 unique OsPsbS1 alleles (on the x-axis; n=1-4 technical replicates each) after 10 minutes of light exposure. Results for each unique allele are plotted, ordered from lowest to highest by average NPQ ability (left to right). Nipponbare WT NPQ (darkest grey, open circles; far right) is shown for 64 biological replicates across all phenotyping experiments, with boundaries delineated as ±2STD (red, dotted lines). All lines with biological replicates below / above the WT boundary are grouped by phenotype, from left to right: knockout (dark grey; circles), knockdown (dark grey; squares), WT-like (grey; hexagons), and overexpression (light grey; diamonds). Figure 3B The proportion of each phenotype observed is provided, with shading and Figure 3A The same as used in ; knockout = 21.67%, knockdown = 15%, WT-like = 61.67%, and overexpression = 1.67%.

[0020] Figures 4A-4J We show that changes in PsbS protein abundance underlie the NPQ and ΦPSII phenotypes. Figure 4A The light intensity adaptation scheme used to assess steady-state chlorophyll fluorescence characteristics is shown, starting with 0 μE for 15 min, then 100 μE for 45 min (shown as three 15 min blocks), 800 μE for 45 min (shown as three 15 min blocks), 1500 μE for 45 min (shown as three 15 min blocks), and ending with 0 μE for 15 min. For each light intensity, the steady-state chlorophyll fluorescence values ​​of the last 5 measurements (spanning 15 min) were averaged for data visualization (indicated by 5 black triangles). Initial F v / F m and F after high light (HL) v / F m The measured values ​​are indicated by individual black triangles at the beginning and end of the schematic (n=3-4 biological replicates, plotted above the mean±1 SEM). Figure 4B The initial F values ​​of representative lines from events 2 and 19 are shown. v / F m (On the y-axis), from left to right: Event 2 Nipponbare WT control (black, hollow circles, Event 2_WT), Event 2 knockout (dark gray, circle, 2-5_KO), Event 2 strong knockdown (dark gray, square, 2-1_SKD), Event 2 weak knockdown (gray, triangle, 2-6_WKD), Event 2 WT-like (gray, hexagon, 2-4_WT-like), Event 2 overexpression (light gray, diamond, 2-4_OX), Event 19 Nipponbare WT control (black, hollow circles, Event 19_WT), Event 19-1 WT-like (dark gray, hexagon, 19-1_WT-like) and Event 19-1 overexpression (dark gray, diamond, 19-1_OX). Figure 4C Showing 100, 800 and 1500 μmol m -2 s -1The difference in steady-state NPQ capacity at . Event 2 strains are shown on the left side of the dotted line: event 2 Nipponbare WT control (black, open circles, event 2_WT), event 2 knockout (dark gray, circle, 2-5_KO), event 2 strong knockdown (dark gray, square, 2-1_SKD), event 2 weak knockdown (gray, triangle, 2-6_WKD), event 2 WT-like (gray, hexagon, 2-4_WT-like) and event 2 overexpression (light gray, diamond, 2-4_OX). Event 19 strains are shown on the right side of the dotted line: event 19 Nipponbare WT control (black, open circles, event 19_WT), event 19-1 WT-like (dark gray, hexagon, 19-1_WT-like) and event 19-1 overexpression (dark gray, diamond, 19-1_OX). Figure 4D Shown are immunoblots for OsPsbS1 of representative event 2 lines (from left to right: 2-5KO, 2-1SKD, 2-6WKD, 2-4WT-like, and 2-4OX) compared to event 2WT (far left). Figure 4E Shown are immunoblots of OsPsbS1 of representative event 2 lines (from left to right: 2-1SKD, 2-6WKD, and 2-4OX) compared to varying amounts of event 2 WT protein (far left and far right: 1 / 8, 1 / 4, 1 / 2, 1x, 2x, and 4x of 5 μg protein). Figure 4F Immunoblots of OsPsbS1 of representative event 19 lines (19-1OX and 19-1WT-like) are shown compared to event 19WT (far left). Figures 4D-4F Unless otherwise stated, 5 μg of total protein was loaded per well, and representative blots of two technical replicates are shown. Figure 4G Shown is 1500 μmol m plotted against OsPsbS1 band density (on the x-axis). -2 s -1NPQ capacity at (on the y-axis), which is normalized to 1) Atpβ band intensity and 2) WT PsbS band intensity on the same blot (PsbS / Atpβ normalized to WT=1). Event 2 and event 19 strains are shown: event 2 Nipponbare WT control (black, open circles, event 2_WT), event 2 knockout (dark gray, circle, 2-5_KO), event 2 strong knockdown (dark gray, square, 2-1_SKD), event 2 weak knockdown (gray, triangle, 2-6_WKD), event 2 WT-like (gray, hexagon, 2-4_WT-like), event 2 overexpression (light gray, diamond, 2-4_OX), event 19 Nipponbare WT control (black, open circles, event 19_WT), event 19-1 WT-like (dark gray, hexagon, 19-1_WT-like) and event 19-1 overexpression (dark gray, diamond, 19-1_OX). Data were fit to a logarithmic curve using each individual data point (mean ± 1 SEM shown), with dashed lines limiting 95% confidence intervals. Figure 4H The correlation between NPQ and ΦPSII for lines with WT or higher NPQ capacity (e.g., OX lines) at all three light intensities is shown, represented by colored circles (light gray, 100 μmol m -2 s -1 ; Gray, 800 μmol m -2 s -1 ; Dark grey, 1500 μmol m -2 s -1 ) and the fitted linear regression, where the dashed lines limit the 95% confidence interval. Fig. 4I The residual F is shown 15 minutes after the end of the increasing light intensity protocol. v / F m (n = 3-4 biological replicates, plotted above mean ± 1 SEM). Event 2 strains are shown on the left side of the dashed line: event 2 Nipponbare WT control (black, open circles, event 2_WT), event 2 knockout (dark gray, circle, 2-5_KO), event 2 strong knockdown (dark gray, square, 2-1_SKD), event 2 weak knockdown (gray, triangle, 2-6_WKD), event 2 WT-like (gray, hexagon, 2-4_WT-like) and event 2 overexpression (light gray, diamond, 2-4_OX). Event 19 strains are shown on the right side of the dashed line: event 19 Nipponbare WT control (black, open circles, event 19_WT), event 19-1 WT-like (dark gray, hexagon, 19-1_WT-like) and event 19-1 overexpression (dark gray, diamond, 19-1_OX). Figure 4J The event 2 strains were shown to be -2 s -1 Residue F v / F mLinear regression for NPQ capacity: event 2 Nipponbare WT control (black, open circles, event 2_WT), event 2 knockout (dark grey, circles, 2-5_KO), event 2 strong knockdown (dark grey, squares, 2-1_SKD), event 2 weak knockdown (grey, triangles, 2-6_WKD), event 2 WT-like (grey, hexagons, 2-4_WT-like) and event 2 overexpression (light grey, diamonds, 2-4_OX). For all pairwise comparisons, significance was determined by ordinary one-way ANOVA (α=0.05) using Dunnett's test for Nipponbare WT ( Figure 4B-4C ) or the respective event 2 or event 19 overexpression datasets ( Fig. 4I ) were performed for multiple comparisons and are indicated by asterisks (**p≤0.01, ***p≤0.001, ****p<0.0001).

[0021] Figures 5A-5E Shown are chlorophyll fluorescence and gas exchange measurements for event 2 lines with varying PsbS abundance. Figure 5A The NPQ (on the y-axis) is shown relative to different red light intensities (on the x-axis) in μE. Figure 5B The operating efficiency of PSII (ΦPSII; on the y-axis) is shown relative to different red light intensities (on the x-axis) in μE. Figure 5C CO is shown relative to different red light intensities (on the x-axis) in μE. 2 Assimilation (A n (μmol m -2 s -1 ); on the y-axis). Figure 5D The stomatal conductance (g) is shown relative to different red light intensities (on the x-axis) in μE. sw (mol H 2 O m -2 s -1 ); on the y-axis). Figures 5A-5D Measurements were made as a function of incident red light on mature flag leaves (n = 4 biological replicates, data are shown as ± 1 SEM), and significance was determined by two-way ANOVA (α = 0.05) using Dunnett's test for multiple comparisons against Nipponbare WT control and is indicated by asterisks (**p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001). Figure 5E The iWUE (μmol CO 2 mol -1 H 2 O; on the y axis)(A n / g sw) Linear regression as a function of the maximum NPQ capacity (2000uE; on the x-axis) of each genotype (n=4 biological replicates, individual data points and mean±1SEM are shown). Figures 5A-5E In the figure, the genotypes are marked as follows: Nipponbare WT control (black, open circles, Nip_WT), event 2 strong knockdown (dark grey, squares, 2-1_SKD), event 2 weak knockdown (grey, triangles, 2-6_WKD), event 2 WT-like 1 (grey, hexagons, 2-4_WT-like), event 2 WT-like 2 (dark grey, inverted triangles, 2-7_WT-like), and event 2 overexpression (light grey, diamonds, 2-4_OX).

[0022] Figures 6A-6D Shows Q A Redox state (1-qL) and g sw Relevance of PsbS as a predictor of iWUE with varying levels of PsbS. Fig. 6A Shows Q A Redox state (1-qL; on the y-axis) as a function of incident red light (on the x-axis) on mature flag leaves (n=4 biological replicates, data shown as ± 1 SEM). Significance was determined by two-way ANOVA (α=0.05) using Dunnett's test for multiple comparisons against Nipponbare WT control. Figure 6B The 1-qL (on the x-axis) of all genotypes (n=24 biological replicates; data are shown as ±1 SEM) is shown as g sw (mol H 2 O m -2 s -1 )(on the y-axis). Figure 6C In all cases above 50 μmol m -2 s -1 In the light step, 1-qL (on the x-axis) is g sw (mol H 2 O m -2 s -1 Linear regression of the function of ) (on the y-axis) (n=4 biological replicates each) (data are shown as ±1SEM). For each genotype, r 2 The values ​​are shown in the table below the graph: 2-1SKD = ​​0.9766; 2-4OX = 0.9831; 2-4WT = 0.9866; 2-6WKD = 0.9974; 2-7WT = 0.9945; NipWT = 0.9744. Fig.6D The slopes (left) and y-intercepts (right) of the linear regression lines by genotype are shown (data shown as ± 1 SEM). Fig.6DPairwise significance in was determined by ordinary one-way ANOVA (α=0.05) using Dunnett's test for multiple comparisons against Nipponbare WT and is indicated by asterisks (**p≤0.01, ***p≤0.001, ****p<0.0001). Fig. 6A , 6C and 6D, the genotypes are marked as follows: Nipponbare WT control (black, open circles, Nip_WT), event 2 strong knockdown (dark gray, squares, 2-1_SKD), event 2 weak knockdown (gray, triangles, 2-6_WKD), event 2 WT-like 1 (gray, hexagons, 2-4_WT-like), event 2 WT-like 2 (dark gray, inverted triangles, 2-7_WT-like), and event 2 overexpression (light gray, diamonds, 2-4_OX).

[0023] Figures 7A-7C These results suggest that variation near and within the 5'UTR is likely driving the observed phenotypic variation. Fig. 7A Three unique cis-regulatory mutants with large deletions (black links) mapped to the Nipponbare (ssp. japonica) promoter at the distal gRNA site (grey triangles) are shown for lines 17-1_7 (top), 25-3_16 (middle), and 25-4_18 (bottom). Scale bar is 250 bp. Figure 7B Shows Fig. 7A NPQ kinetics of the distal deletion lines of the medium and large, as follows: event 17-1_7 (grey circles), event 25-3_16 (grey squares), event 25-4_18 (grey triangles), Nipponbare WT (grey inverted triangles). NPQ (on the y-axis) was measured over time (on the x-axis; 10 minutes in light, then 10 minutes in the dark). Data are shown as ± 1 SEM. Figure 7C Shows Figures 4A-4J , 5A-5E and 6A-6D with different NPQ ability of the strains of the proximal gRNA (grey triangle at the top) PCR genotyping of event 2 mutations. From top to bottom: KO strains, strong KD strains, weak KD strains, WT sample lines, 24-5_18 and Nipponbare WT. Event 2-4_OX cannot be genotyped by PCR. Mutations in the cis-regulated promoter (black box) and 5'UTR (white box) are shown upstream of the OsPsbS1 coding sequence. The dotted line represents the 146bp WT sequence not shown before the start codon. Deletions are shown in dark gray, and insertions are shown in gray. The scale bar is 50bp.

[0024] Figures 8A-8D Long-read sequencing of two overexpressed alleles identified an inversion upstream of OsPsbS1. Fig. 8AShown are increasing resolution dot plots of the chromosome 1 locus (-1.1 Mbp) containing OsPsbS1 for the 2-4_OX line, where breaks in continuity indicate the presence of genomic inversions. Figure 8B Increased resolution of a chromosome 1 locus with a -254 kb inversion (upstream of OsPsbS1 for 2-4_OX line in Integrative Genomics Viewer (IGV), chromosome 1: 37693233-37948089). Figure 8C Shown is a dot plot of increased resolution for the chromosome 1 locus (-1.5 Mbp) containing OsPsbS1 for the 19-1_OX line. Fig.8D Shown is the increased resolution of the chromosome 1 locus with an inversion ~3-4 kb upstream of OsPsbS1 (chromosome 1: ~37693800–37696800) for the 19-1_OX line in IGV. Figure 8B and 8D In the figure, the gray box indicates the OsPsbS1 gene (LOC_Os01g64960) (in Figure 8B The upper left, and Fig.8D The strength of the called structural variants was quantified by Sniffles (top grey arrow, quantified in dark grey) and confirmed by head-to-head sequencing of the fragments (bottom grey arrow).

[0025] Figures 9A-9D It was shown that the overexpression phenotype is constitutive, but OsPsbS1 expression is not constitutive. Fig. 9A Shown are qPCR results from one time point comparing OsPsbS1 expression in the overexpression line (2-4_OX), the wild-type line (WT), and the knockout line (2-5_KO). Fig. 9B Shown are qPCR results from one time point comparing OsPsbS1 expression in the overexpression line (2-4_OX), wild-type line (WT), wild-type-like line (2-4_WT-like), weak knockout line (2-6_WKD), strong knockout line (2-1_SKD), and knockout line (2-5_KO). Fig. 9C Shown are the fold changes in OsPsbS1 expression in the wild-type line (WT) compared to the overexpression line (2-4_OX) over a time course, where plants were grown in 14-hour day length (DL). OsPsbS1 transcript levels in 2-4_OX were statistically significantly higher only at the first time point (p<0.01). Fig.9DIt is shown that over the course of time, time of day had negligible effect on NPQ in wild-type lines (WT), strong knockdown lines (2-1_SKD), overexpression lines (2-4_OX), and weak knockdown lines (2-6_WKD), where plants were grown in a 14-hour day length (DL). Details

[0026] The following description sets forth exemplary methods, parameters, etc. However, it should be appreciated that such description is not intended as a limitation on the scope of the present disclosure, but is provided as a description of exemplary embodiments. Screening methods for gain-of-function mutations

[0027] Aspects of the present disclosure include methods for screening a target gene of a plant for a gain-of-function mutation, the method comprising: (a) generating a set of mutations in a non-coding sequence (NCS) of a target gene in a plant cell population of a plant using one or more RNA-guided nucleic acid modification enzymes targeting the target gene, wherein the one or more RNA-guided nucleic acid modification enzymes targeting the target gene include one or more different guide RNAs; (b) regenerating the plant cell population into two or more plants that are hemizygous for the generated mutations; (c) (1) selfing the two or more plants to produce progeny plants, and (2) optionally, screening the progeny plants that are homozygous for the mutation for screening in part (d); and (d) screening the progeny plants from step (c) to identify gain-of-function mutations. Additional embodiments of this aspect further include: (e) selecting plants with gain-of-function mutations, and (f) sequencing the target gene to determine the gain-of-function mutations. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the one or more RNA-guided nucleic acid modification enzymes are expressed by an expression vector that includes a selectable marker, and the screening in step (d) includes screening plants that lack the selectable marker. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the gain-of-function mutation induces overexpression of the target gene. In a further embodiment of this aspect, the overexpression of the target gene is in the morning. In another embodiment of this aspect, the overexpression of the target gene is not constitutive. In still another embodiment of this aspect, the plant has a constitutive phenotype. In yet another embodiment of this aspect (which may be combined with any of the preceding embodiments), the target gene induction is related to photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield and CO 2One or more relevant phenotypes in fixation, and the screening of step (c) (2) includes screening offspring plants by chlorophyll fluorescence to identify non-transgenic plants that are assumed to be homozygous for mutations. In this respect, in still another embodiment (which can be combined with any of the aforementioned embodiments), the target gene induces a phenotype related to water use efficiency, and the screening of step (c) (2) includes screening offspring plants by chlorophyll fluorescence to identify non-transgenic plants that are assumed to be homozygous for mutations. Chlorophyll fluorescence measurement phiPSII can be used to monitor photosynthetic efficiency under illumination. When one or more RNA-guided nucleic acid modification enzymes are connected to an antibiotic resistance marker (such as hygromycin), antibiotic sensitivity screening (such as hygromycin sensitivity screening) can be used to increase the separation and / or loss flux of screening one or more RNA-guided nucleic acid modification enzymes. The sensitivity of this method is significantly cheaper and more effective than PCR in some plant species (such as rice) because PCR may be susceptible to contamination and false positive / negative effects. By combining this method with the screening of the desired phenotype, it is possible to improve the screening flux and more quickly identify the strains that are homozygous for the mutation of interest. In still another embodiment of this aspect (which may be combined with any of the preceding embodiments), the method does not include the use of plants having hypomorphic or null alleles of the target gene. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the gain-of-function mutation improves the yield, quality, or both of the plant having the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the one or more RNA-guided nucleic acid modifying enzymes targeting the target gene include two or more different guide RNAs, three or more different guide RNAs, four or more different guide RNAs, five or more different guide RNAs, ten or more different guide RNAs, or twenty or more different guide RNAs (e.g., twenty-four different guide RNAs). In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the one or more different guide RNAs may be dispersed in multiple boxes within a single T-DNA insertion (e.g., 3 boxes, each with 8 gRNAs).

[0028] In yet another embodiment of this aspect (which may be combined with any of the foregoing embodiments), each guide RNA targets a region of the target gene selected from the following: a promoter region, an upstream regulatory region, a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), an intron, a micro-RNA binding site (e.g., a micro-RNA binding site in an mRNA, which may be located in a coding region), an alternative splicing element, and a downstream regulatory element. In a further embodiment of this aspect, the guide RNA targets the 5'UTR of the target gene. Studies in corn have shown that 5'UTR may be the best source of natural allelic variation in protein expression (Gage et al., (2022) Variation in upstream open reading frames contributes to allelic diversity in maize protein abundance. PNAS, 119 (14) e2112516119). In addition, editing a small region (e.g., 5'UTR) can provide a dynamic range of expression from strong knockdown to overexpression. In another embodiment of this aspect (which can be combined with any of the preceding embodiments), the guide RNA targets at least one region in the target gene that is at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical across plant species. In still another embodiment of this aspect (which can be combined with any of the preceding embodiments), at least 50% of the mutation group is located in a region of the target gene selected from the group consisting of a promoter region, an upstream regulatory region, a 5'UTR, a 3'UTR, an intron, a micro-RNA binding site (e.g., a micro-RNA binding site in an mRNA, which can be located in a coding region), an alternative splicing element, and a downstream regulatory element. In a further embodiment of this aspect, at least 50% of the mutation group is located in the 5'UTR of the target gene. In still another embodiment of this aspect (which can be combined with any of the preceding embodiments), the gain-of-function mutation is a deletion, an inversion, a translocation, an insertion, a conversion, a transversion, or a combination thereof. In further embodiments of this aspect, the mutation may change one base or more than one base. In another embodiment of this aspect, the transition or transversion mutation is caused by changing one or more bases in the sequence. In yet another embodiment of this aspect (which may be combined with any of the foregoing embodiments), the gain-of-function mutation is an increase in target gene expression, an increase in target gene transcription, an increase in the stability of mRNA produced by the target gene, an increase in the translation of the mRNA protein coding region, or a decrease in mRNA degradation compared to plants lacking the gain-of-function mutation grown under the same conditions in each case. In another embodiment of this aspect, the gain-of-function mutation is in the morning.In other embodiments of this aspect (which may be combined with any of the foregoing embodiments), one or more RNA-guided nucleic acid modification enzymes are Cas enzymes, base editors, or lead editors. In further embodiments of this aspect, the Cas enzyme is selected from the group consisting of Cas9, Cas12, Cas12a, Cas13, Cas14, CasX, or CasY. In another embodiment of this aspect, the base editor includes a cytidine base editor or an adenine base editor (e.g., a dCas9 fusion protein or a Cas9 nickase fusion protein). In a further embodiment of this aspect, the lead editor includes a catalytically impaired Cas9 endonuclease, such as a Cas9 nickase. Other RNA-guided nucleic acid modification enzymes including other Cas enzyme types known to those skilled in the art may also be used in the methods disclosed herein. It is believed that when using, for example, Cas9, off-target effects are rare and / or not important in plants. By ensuring that the mutation of interest is 100% separated from the desired phenotype, off-target effects can be excluded because unconnected off-target mutations that work in trans will be separated independently. In some embodiments, targeted editing (e.g., using primer editing) is used to introduce sets of mutations, which can be more efficient than Cas9-directed editing.

[0029] In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with Crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a maize plant, a biomass plant, an Arabidopsis plant, a tobacco (Nicotiana tabacum) plant, a rice (Oryza sativa) plant, a corn (Zea mays) plant, a sorghum (Sorghum bicolor) (sweet sorghum or grain sorghum) plant, a soybean (Glycine max) plant, a cowpea (Vigna unguiculata) plant, a poplar (Populus spp.) plant, a eucalyptus (Eucalyptus spp.) plant, a cassava (Manihot esculenta) plant, a barley (Hordeum vulgare) plant, a potato (Solanum tuberosum plants, sugarcane (Saccharum spp.) plants, alfalfa (Medicago sativa) plants, Miscanthus plants, energy sugarcane plants, elephant grass plants, wheat plants, oat plants, oil palm plants, safflower plants, sesame plants, flax plants, cotton plants, sunflower plants, camelina plants, rapeseed (Brassicanapus) plants, Ethiopian mustard (Brassica carinata) plants, mustard (Brassica juncea) plants, pearl millet plants, millet plants, other cereal plants, oilseed plants, vegetable crop plants, forage crop plants, industrial crop plants or woody crop plants. In another embodiment of this aspect, the plant is a rice (Oryza sativa) plant, a corn (Zeamays) plant or a cowpea (Vigna unguiculata) plant. In a further embodiment of this aspect (which can be combined with any of the preceding embodiments), the plant is an elite line or an elite strain.

[0030] In a further embodiment of this aspect (which may be combined with any of the preceding embodiments), the target gene is selected from the group consisting of a photosystem II subunit S (PsbS) gene, a zeaxanthin epoxidase (ZEP) gene, and a violaxanthin de-epoxidase (VDE) gene. In an additional embodiment of this aspect, the screening comprises evaluating one or more of the following: photosynthetic efficiency under fluctuating light conditions; photoprotection efficiency under fluctuating light conditions; increase in non-photochemical quenching (NPQ) induction rate under fluctuating light conditions; increase in non-photochemical quenching (NPQ) relaxation rate under fluctuating light conditions; improvement in quantum yield under fluctuating light conditions, and CO under fluctuating light conditions. 2Improved fixation. In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a PsbS gene, and wherein the PsbS gene comprises a sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:97 or SEQ ID NO:98. In yet another embodiment of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a ZEP gene, and the ZEP gene comprises SEQ ID NO:92. In further embodiments of this aspect (which can be combined with any of the preceding embodiments), wherein the target gene is selected from PsbS, ZEP or VDE, the target gene is a VDE gene, and wherein the VDE gene comprises a sequence selected from the group consisting of SEQ ID NO:92 or SEQ ID NO:95. In yet another embodiment of this aspect, the one or more different guide RNAs include a spacer sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.In still another embodiment of this aspect, one or more different guide RNAs include a spacer sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In a further embodiment of this aspect, one or more different guide RNAs include a spacer sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65. In yet another embodiment of this aspect (which can be combined with any of the aforementioned embodiments having an intervening sequence), a vector is used to introduce one or more different guide RNAs, and wherein the vector comprises two or more gRNA scaffolds, wherein the gRNA scaffold comprises SEQ ID NO: 9 and two or more tRNA linkers having SEQ ID NO: 10. Methods for producing improved cash crop plants or crop seeds and improved plants

[0031] Some aspects of the present disclosure relate to methods for producing improved cash crop plants or crop seeds, the methods comprising: (a) selecting cash crop plants for improvement; (b) introducing a gain-of-function mutation identified in any of the methods of the foregoing embodiments into at least one cash crop cell to produce an improved cash crop cell; and (c) producing improved cash crop plants or crop seeds from the improved cash crop plant cells. In further embodiments of this aspect, the cash crop plants include rice (Oryza sativa) plants, corn (Zea mays) plants, or cowpea (Vigna unguiculata) plants.

[0032] Additional aspects of the present disclosure include improved economic crop plants or crop seeds comprising a gain-of-function mutation in a non-coding sequence of a target gene, wherein the target gene induces photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO 2The invention relates to a phenotype related to one or more of fixation and water use efficiency, and wherein the gain-of-function mutation improves the yield, quality, or both of the plant with the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions. Further embodiments of this aspect include cash crop plants, including rice (Oryza sativa) plants, corn (Zea mays) plants, or cowpea (Vigna unguiculata) plants. In another embodiment of this aspect (which can be combined with any of the foregoing embodiments), the target gene is selected from the photosystem II subunit S (PsbS) gene, zeaxanthin epoxidase (ZEP) gene, and violaxanthin de-epoxidase (VDE) gene. Further aspects of the present application relate to plant parts, tissues, or cells of the plants of any of the foregoing embodiments.

[0033] Further aspects of the present disclosure include improved plants comprising an inversion in a PSBS gene cis-regulatory element, wherein the inversion increases PsbS gene expression. In a further embodiment of this aspect, the plant is a rice (Oryza sativa) plant. In still another embodiment of this aspect, the rice plant is a japonica rice subspecies (Oryza sativa ssp. japonica) plant. In yet another embodiment of this aspect (which may be combined with any of the preceding embodiments), the increased PsbS gene expression compared to a control plant that does not have an inversion in a PsbS gene cis-regulatory element includes overexpression, increased expression at one or more specific times, increased expression in one or more specific tissues, increased expression at one or more developmental stages, or a combination thereof. In still another embodiment of this aspect (which may be combined with any of the preceding embodiments), the inversion encompasses a portion of the 5'UTR of the PsbS gene. In another embodiment of this aspect, which may be combined with any of the preceding embodiments, the inversion comprises between 1,000 and 500,000 nucleotides, between 2,000 and 400,000 nucleotides, between 3,000 and 300,000 nucleotides, between 1,000 and 10,000 nucleotides, between 2,000 and 8,000 nucleotides, between 3,000 and 5,000 nucleotides, or about 4,000 nucleotides. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant or its ancestor is screened for inversions in the cis-regulatory element of the PsbS gene and increased expression of the PsbS gene. In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the inversion in the cis-regulatory element of the PsbS gene is randomly generated in the plant or its ancestor. In still another embodiment of this aspect, the inversion is randomly generated using a guide RNA.In a further embodiment of this aspect, the guide RNA comprises a spacer sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased PsbS gene expression results in photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO 2 In another embodiment of this aspect, the increased PsbS gene expression comprises overexpression of PsbS in the morning, or is not constitutive overexpression.

[0034] "Control plants" as described herein can be control samples or reference samples from wild-type, heterozygous or ineffective segregating plants, species or samples, or from populations thereof. Reference values ​​can be used to replace control samples or reference samples previously obtained from wild-type, heterozygous or ineffective segregating plants, species or samples, or from populations thereof or a group of wild-type, heterozygous or ineffective segregating plants, species or samples. Control samples or reference samples can also be samples with known amounts of detectable compositions or spiked samples.

[0035] The term "plant" is used in its broadest sense. It includes, but is not limited to, any kind of woody plants, ornamental or decorative plants, crop or cereal plants, fruit or vegetable plants and algae (e.g., Chlamydomonas reinhardtii). It also refers to a large number of plant cells that are largely differentiated into structures present at any stage of plant development. Such structures include, but are not limited to, fruits, buds, stems, leaves, petals, etc.

[0036] The term "plant tissue" includes plant differentiated and undifferentiated tissues, including tissues present in roots, shoots, leaves, inflorescences, anthers, pollen, ovaries, seeds, and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissues may be present in plantations, organ cultures, tissue cultures, or cell cultures.

[0037] The term "plant part" as used herein refers to a plant structure, plant organ or plant tissue. In some embodiments, a plant part can be a seed, pod, fruit, leaf, flower, stem, root, any part of the foregoing or a cell thereof, or a non-renewable part or cell of a genetically modified or improved plant part. As used herein, a "non-renewable" part or cell of a genetically modified or improved plant or part thereof is a part or cell of a whole plant that itself cannot be induced to form a whole plant or cannot be induced to form a whole plant that can carry out sexual and / or asexual reproduction. In some embodiments, a non-renewable part or cell of a plant part is a transgenic seed, pod, fruit, leaf, flower, stem or root part or a cell thereof.

[0038] Also provided are processed plant products, which include detectable amounts of nucleotide fragments, expressed RNA and / or proteins comprising the genetic modification disclosed herein. Such processed products include, but are not limited to, plant biomass, oil, meal, animal feed, flour, flakes, bran, cotton wool, shells and processed seeds. The processed product may be non-renewable. Plant products may include commodities or other commercial products derived from transgenic plants or transgenic plant parts, wherein commodities or other products in commerce may be tracked by detecting nucleotide fragments, expressed RNA and / or proteins comprising unique portions of the genetic modification disclosed herein.

[0039] The term "plant cell" refers to the structural, physiological unit of a plant, including a protoplast and a cell wall. Plant cells may exist as isolated single cells or cultured cells, or as part of a higher organizational unit such as, for example, a plant tissue, a plant organ, or a whole plant. The term "plant cell culture" refers to the culture of plant units such as, for example, protoplasts, cells and cell clusters in liquid culture medium or on solid culture medium, cells in plant tissues and organs, microspores and pollen, pollen tubes, anthers, ovules, embryo sacs, fertilized eggs, and embryos at different stages of development.

[0040] The term "plant material" refers to leaves, stems, roots, inflorescences and flowers or flower parts, fruits, pollen, anthers, egg cells, fertilized eggs, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0041] "Plant organ" refers to a distinct and visible structure and differentiated part of a plant, such as a root, stem, leaf, bud, inflorescence, spikelet, floret, seed, or embryo.

[0042] The term "crop plants" refers in particular to monocotyledonous plants, such as cereals (wheat, millet, sorghum, rye, triticale, oats, barley, teff, spelt, buckwheat, fonio and quinoa), rice, maize (corn) and / or sugar cane; or dicotyledonous crops, such as beets (such as sugar beet or fodder beet); fruits (such as pome, stone fruit or soft fruit, for example apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries or blackberries); leguminous plants (such as beans, lentils, peas or soybeans); oilseed plants (such as rapeseed, mustard, poppy, olive, The present invention relates to plants such as sunflower, coconut, castor oil plant, cocoa bean or peanut); cucumber plants (such as zucchini, cucumber or melon); fiber plants (such as cotton, flax, hemp or jute); citrus fruits (such as orange, lemon, grapefruit or mandarin); vegetables (such as spinach, lettuce, cabbage, carrot, tomato, potato, gourd or red pepper); lauraceae (such as avocado, cinnamon or camphor); tobacco; nuts; coffee; tea; grapevine; hops; durian; banana; natural rubber plant; and ornamental plants (such as flowers, shrubs, broadleaf trees or evergreen trees, such as conifers). This list does not represent any limitation.

[0043] The term "woody crop" or "woody plant" refers to a plant that produces wood as its structural tissue. Woody crops include trees, shrubs or vines. Examples of woody crops include, but are not limited to, Robinia pseudoacacia, hybrid chestnut, black walnut, Japanese maple, eucalyptus, Casuarina, spruce, fir, pine and dogwood.

[0044] The term "improved growth" or "increased growth" is used herein in its broadest sense. It includes any improvement or enhancement in plant growth and development processes. Examples of improved growth include, but are not limited to, increased photosynthetic efficiency, increased biomass, increased yield, increased seed number, increased seed weight, increased stem height, increased leaf area, increased root biomass, and increased plant dry weight.

[0045] The term "quantum yield" refers to the amount of CO fixed per mole of absorbed photons. 2 moles of CO 2 The efficiency of converting light into fixed carbon. The quantum yield of photosynthesis is derived from the measurement of light intensity and photosynthetic rate. Therefore, quantum yield is a measure of the efficiency of absorbed light to produce a specific effect. The amount of photosynthesis carried out in plant cells or plants can be indirectly detected by measuring the amount of starch produced by transgenic plants or plant cells. CO can also be used 2 Detectors (e.g., CO 2 A decrease or consumption of O indicates an increase in photosynthetic levels) or 2 Detector (e.g., O 2 The amount of photosynthesis in plant cell culture or plants can be detected by measuring the amount of photosynthesis in plant cell culture or plants (see, for example, Silva et al., Aquatic Biology 7:127-141, 2009; and Bai et al., Biotechnol. Lett. 33:1675-1681, 2011). Radiolabeled CO can also be used. 2 (For example, 14CO 2 and H 14 CO 3 - ) to measure photosynthesis (see, e.g., Silva et al., Aquatic Biology 7:127-141, 2009, and methods described in references cited therein). Photosynthesis can also be measured by detecting chlorophyll fluorescence (e.g., Silva et al., Aquatic Biology 7:127-141, 2009, and references cited therein). Other methods for detecting photosynthesis in plants are described in Zhang et al. Mol. Biol. Rep. 38:4369-4379, 2011.

[0046] In the natural sciences, the term "relaxation" refers to the return of a perturbed system to equilibrium, typically from a high energy level to a low energy level. As used herein, the term "non-photochemical quenching relaxation" or "NPQ relaxation" refers to the process by which NPQ levels decrease when transitioning from high to low light intensities.

[0047] References herein to "about" values ​​or parameters refer to the usual error ranges for the corresponding values ​​that are readily known to those skilled in the art. References herein to "about" values ​​or parameters include (and describe) aspects related to the value or parameter itself. For example, references to descriptions of "about X" include descriptions of "X". Molecular biological methods for producing transgenic plants, plant parts and plant cells

[0048] One aspect of the present disclosure provides transgenic plants, plant parts or plant cells comprising a gain-of-function mutation in a non-coding sequence of a target gene, including photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP) and violaxanthin de-epoxidase (VDE). In addition, the present disclosure provides guide RNA (gRNA) spacer sequences, gRNA scaffold sequences and tRNA linker sequences, which can be used to screen and / or generate mutations in the non-coding sequence of a target gene in a plant cell population.

[0049] Transformation and generation of genetically altered monocot and dicot cells are well known in the art. See, for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed.: Gartland, Humana Press Inc. (1995); Wang et al., Acta Hort. 461:401-408 (1998), and Broothaerts et al., Nature 433:629-633 (2005). The choice of method varies with the type of plant to be transformed, the specific application, and / or the desired result. The skilled practitioner will readily select an appropriate transformation technique.

[0050] Any known method for deleting, inserting or otherwise modifying cell DNA (e.g., genomic DNA and organelle DNA) in the art can be used to practice the compositions, methods and processes disclosed herein. For example, CRISPR / Cas-9 systems and related systems (e.g., TALEN, ZFN, ODN, etc.) can be used to insert heterologous genes into target sites in genomic DNA, or substantially edit endogenous genes to express heterologous genes, or modify promoters to increase or otherwise change the expression of endogenous genes, such as by removing repressor binding sites or introducing enhancer binding sites. For example, a disarmed Ti plasmid containing a gene construct for deleting or inserting a target gene in Agrobacterium can be used to transform plant cells, and thereafter, the method described in the art (e.g., in EP 0116718, EP 0270822, PCT Publication WO 84 / 02913 and published European Patent Application ("EP") 0242246) can be used to regenerate transformed plants from transformed plant cells. The Ti plasmid vectors each contain genes between the border sequences of the T-DNA of the Ti plasmid, or at least located to the left of the right border sequence. Of course, other types of vectors can be used to transform plant cells, using methods such as direct gene transfer (e.g., as described in EP 0233247), pollen-mediated transformation (e.g., as described in EP 0270356, PCT publication WO 85 / 01856 and U.S. Patent No. 4,684,611), plant RNA virus-mediated transformation (e.g., as described in EP 0 067 553 and U.S. Patent No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Patent No. 4,536,475) and other methods, such as for transforming corn (U.S. Patent No. 6,140,553; Fromm et al., Bio / Technology (1990) 8,833-839); Gordon-Kamm et al., The Plant Cell, (1990) 2, 603-618)), methods for transforming certain strains of rice (Shimamoto et al., Nature, (1989) 338, 274-276; Datta et al., Bio / Technology, (1990) 8, 736-740)), and methods for transforming monocotyledons in general (PCT publication WO 92 / 09696). For cotton transformation, the methods described in PCT patent publication WO 00 / 71733 can be used. For soybean transformation, reference is made to methods known in the art, such as the methods of Hinchee et al. (Bio / Technology, (1988) 6, 915) and Christou et al. (Trends Biotech, (1990) 8, 145) or WO 00 / 42207.

[0051] The genetically modified plants of the present disclosure can be used in conventional plant breeding programs to produce more genetically modified plants with the same characteristics, or to introduce genetic changes into other varieties of the same or related plant species. The seeds obtained from the modified plants preferably contain the genetic changes as stable inserts in the chromosomal DNA or as modifications to endogenous genes or promoters. Plants comprising genetic changes according to the present disclosure include plants, such as fruit trees or ornamental plants, containing or derived from rootstocks of plants comprising genetic changes of the present disclosure. Therefore, any non-transgenic grafted plant parts on the inserted transformed plants or plant parts are included in the present disclosure.

[0052] The genetic alterations of the present disclosure included in expression vectors or expression cassettes (which result in expression of an introduced gene or altered expression of an endogenous gene) will generally utilize plant-expressible promoters. As used herein, "plant-expressible promoter" refers to a promoter that ensures expression of the genetic alterations of the present disclosure in plant cells. Examples of constitutive promoters commonly used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter (KAY et al., Science, 236, 4805, 1987), the minimal CaMV 35S promoter (Benfey & Chua, Science, (1990) 250, 959-966), various other derivatives of the CaMV 35S promoter, the figwort mosaic virus (FMV) promoter (Richins et al., Nucleic Acids Res. (1987) 15: 8451-8466), the maize ubiquitin promoter (CHRISTENSEN & QUAIL, Transgenic Res, 5, 213-8, 1996), the clover promoter (Ljubql, MAEKAWA et al., Mol Plant Microbe Interact. 21, 375-82, 2008), the vein mosaic virus promoter (International Application WO 97 / 48819) and Arabidopsis UBQ10 promoter (Norris et al., Plant Mol. Biol. 21, 895-906, 1993).

[0053] Additional examples of promoters that direct constitutive expression in plants are known in the art and include, for example, the strong constitutive 35S promoter ("35S promoter") of cauliflower mosaic virus (CaMV) from isolates CM 1841 (Gardner et al., Nucleic Acids Res, (1981) 9, 2871-2887), CabbB S (Franck et al., Cell (1980) 21, 285-294), and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., Plant Mol Biol, (1992) 18, 675-689), the gos2 promoter (de Pater et al., The Plant J (1992) 2, 834-844), the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581-588), actin promoter (such as the promoter described by An et al. (The Plant J, (1996) 10, 107)), rice actin promoter described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); the promoter of figwort mosaic virus (FMV) (Richins et al., Nucleic Acids Res. (1987) 15: 8451-8466), the promoter of cassava vein mosaic virus (WO 97 / 48819; Verdaguer et al., Plant Mol Biol, (1998) 37, 1055-1067), the pPLEX series promoter from ground clover stunt virus (WO 96 / 06932, in particular the S4 or S7 promoter), alcohol dehydrogenase promoters (e.g., pAdh1S (GenBank Accession Nos. X04049, X00581)), and the TR1' and TR2' promoters driving gene expression 1' and 2' of the T DNA, respectively ("TR1' promoter" and "TR2' promoter", respectively) (Velten et al., EMBO J, (1984) 3, 2723-2730).

[0054] Alternatively, the plant expressible promoter can be a tissue-specific promoter, i.e., a promoter that directs higher levels of expression in certain cells or tissues of the plant (e.g., in green tissues) (such as the promoter of chlorophyll a / b binding protein (Cab)). The plant Cab promoter (Mitra et al., Planta, (2009) 5: 1015-1022) has been described as a strong bidirectional promoter expressed in green tissues (e.g., leaves and stems), and is useful in one embodiment of the present disclosure. These plant expressible promoters can be combined with enhancer elements, they can be combined with minimal promoter elements, or can include repeat elements to ensure the desired expression profile.

[0055] Additional non-limiting examples of tissue-specific promoters include the maize allothionine promoter (DEFRAMOND et al., FEBS 290, 103-106, 1991; application EP 452269), the chitinase promoter (SAMAC et al., Plant Physiol 93, 907-914, 1990), the maize ZRP2 promoter (U.S. Pat. No. 5,633,363), the tomato LeExt1 promoter (Bucher et al., Plant Physiol. 128, 911-923, 2002), the glutamine synthetase soybean root promoter (HIREL et al., Plant Mol. Biol. 20, 207-218, 1992), the RCC3 promoter (PCT application WO 2009 / 016104), the rice anti-ubiquitin promoter (PCT application WO 2007 / 076115), LRR receptor kinase promoter (PCT application WO 02 / 46439) and Arabidopsis pCO2 promoter (HEIDSTRA et al., Genes Dev. 18, 1964-1969, 2004). Further non-limiting examples of tissue-specific promoters include RbcS2B promoter, RbcS1B promoter, RbcS3B promoter, LHB1B1 promoter, LHB1B2 promoter, cab1 promoter, and other promoters described in Engler et al., ACSSynthetic Biology, DOI: 10.1021 / sb4001504, 2014. These plant promoters can be combined with enhancer elements, they can be combined with minimal promoter elements, or can include repeat elements to ensure the desired expression profile.

[0056] In some embodiments, further genetic alterations may be used to increase expression in plant cells. For example, introns at the 5' or 3' ends of an introduced gene, or introns in the coding sequence of an introduced gene, such as the hsp70 intron. Other such genetic elements may include, but are not limited to, promoter enhancer elements, repeated or triple promoter regions, 5' leader sequences that are different from another transgenic or from an endogenous (plant host) gene leader sequence, 3' trailer sequences that are different from another transgenic used in the same plant or from an endogenous (plant host) trailer sequence.

[0057] The introduced gene of the present disclosure can be inserted into the host cell DNA so that the inserted gene portion is located upstream (i.e. 5') of the appropriate 3' end transcriptional regulatory signal (i.e. transcription formation and polyadenylation signal). This is preferably achieved by inserting the gene into the plant cell genome (nucleus or chloroplast). Preferred polyadenylation and transcription formation signals include those of the nopaline synthase gene (Depicker et al., J.Molec Appl Gen, (1982) 1, 561-573), the octopine synthase gene (Gielen et al., EMBO J, (1984) 3: 835-845), the SCSV or malic enzyme terminator (Schunmann et al., Plant Funct Biol, (2003) 30: 453-460) and the T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981-6998), which serve as 3' non-translated DNA sequences in transformed plant cells. In some embodiments, one or more introduced genes are stably integrated into the nuclear genome. When the nucleic acid sequence remains integrated into the nuclear genome and continuously expressed (i.e., producing detectable mRNA transcripts or proteins) throughout subsequent plant generations, there is stable integration. Stable integration into the nuclear genome can be achieved by any method known in the art (e.g., microprojectile bombardment, Agrobacterium-mediated transformation, CRISPR / Cas9, protoplast electroporation, microinjection, etc.).

[0058] The term recombinant or modified nucleic acid refers to a polynucleotide composed of two originally separate sequence fragments, which are artificially manipulated by genetic engineering technology or chemical synthesis to separate polynucleotide fragments. By doing so, people can connect polynucleotide fragments with desired functions to produce the desired functional combination.

[0059] As used herein, the term "overexpression" refers to an increase in expression (e.g., mRNA, polypeptide, etc.) relative to expression in a wild-type organism (e.g., plant) as a result of genetic modification, and may refer to the expression of a heterologous gene at a sufficient level to achieve a desired result (such as, increased yield). In some embodiments, the increase in expression is a slight increase of about 10% over the expression of the wild type. In some embodiments, the increase in expression is an increase of 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) relative to the expression of the wild type. In some embodiments, endogenous genes are upregulated. In some embodiments, exogenous genes are upregulated due to being expressed. Upregulation of genes in plants can be achieved by any method known in the art, including but not limited to the use of constitutive promoters to which inducible response elements have been added, inducible promoters, high expression promoters to which inducible response elements have been added (e.g., the PsaD promoter), enhancers, transcriptional and / or translational regulatory sequences, codon optimization, modified transcription factors, and / or mutated or modified genes that control the expression of genes that are upregulated in response to stimuli such as cytokinin signaling.

[0060] When recombinant nucleic acid is used to express, clone or replicate a specific sequence, the DNA construct prepared for introduction into a host cell generally includes a replication system (e.g., a vector) recognized by the host, which includes an expected DNA fragment encoding a desired polypeptide, and may also include a transcription and translation initiation regulatory sequence operably connected to the polypeptide encoding fragment. In addition, such constructs may include a cell localization signal (e.g., a plasma membrane localization signal). In a preferred embodiment, such DNA constructs are introduced into the genomic DNA, chloroplast DNA, or mitochondrial DNA of a host cell.

[0061] In some embodiments, non-integrated expression systems can be used to induce expression of one or more introduced genes. The expression system (expression vector) may include, for example, an origin of replication or an autonomous replication sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites (such as ribosome binding sites, RNA splicing sites, polyadenylation sites), a transcription terminator sequence and an mRNA stabilizing sequence. Where appropriate, a signal peptide of a secretory polypeptide from the same or related species may also be included, which allows the protein to pass through and / or remain in the cell membrane, cell wall, or be secreted from the cell.

[0062] The selectable marker that can be used for practicing the method disclosed herein can be a positive selectable marker. Generally, positive selection refers to that the cell of genetic alteration can survive in the presence of toxic substances, as long as the recombinant polynucleotide of interest is present in the intracellular situation. Negative selectable markers and screening markers are also well known in the art and are considered by the present disclosure. Those skilled in the art will recognize that any available related markers can be used for practicing the compositions, methods and techniques disclosed herein.

[0063] Recombinant strains disclosed herein can be screened and molecularly analyzed using nucleic acid hybridization techniques. Hybridization procedures are useful for identifying polynucleotides (such as those modified using the techniques described herein) with sufficient homology to the subject regulatory sequences useful for teaching herein. Specific hybridization techniques are not necessary for the present disclosure. With the improvement of hybridization techniques, those skilled in the art can easily apply them. Hybridization probes can be labeled with any appropriate label known to those skilled in the art. Hybridization conditions and washing conditions, such as temperature and salt concentration, can be changed to change the stringency of the detection threshold. See, for example, Sambrook et al. (1989) (see below) or Ausubel et al. (1995), Current Protocols in Molecular Biology, John Wiley & Sons, NY, for further guidance on hybridization conditions in NY.

[0064] In addition, the polymerase chain reaction (PCR) can be used to screen and molecularly analyze genetically altered strains and to create the desired isolated nucleic acid. PCR is a repetitive, enzymatic, primer synthesis of nucleic acid sequences. This procedure is well known and commonly used by those skilled in the art (see Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230: 1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest, which is flanked by two oligonucleotide primers that hybridize to the opposite strands of the target sequence. The 3' ends of the primers are positioned toward each other. These repeated cycles of thermal denaturation of the template, primer annealing to its complementary sequence, and extension of the annealed primers with DNA polymerase result in amplification of the fragment defined by the 5' end of the PCR primer. Since the extension product of each primer can serve as a template for other primers, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in an exponential accumulation of specific target fragments, up to several million-fold within a few hours. The amplification process can be fully automated by using a thermostable DNA polymerase such as Taq polymerase isolated from the thermophilic bacterium Thermusaquaticus. Other enzymes that can be used are known to those skilled in the art.

[0065] The nucleic acids and proteins disclosed herein may also encompass homologs of specifically disclosed sequences. Homology (e.g., sequence identity) may be 50%-100%. In some cases, this homology is greater than 80%, greater than 85%, greater than 90%, or greater than 95%. Those skilled in the art will readily identify the homology or degree of identity required for any intended use of the sequence. As used herein, the percentage sequence identity of two nucleic acids is determined using algorithms known in the art, such as those disclosed in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified as Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into BLASTN, BLASTP, and BLASTX, programs of Altschul et al., (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed using the BLASTN program with score = 100 and word length = 12 to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al., (1997) Nucl. Acids. Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (BLASTN and BLASTX) are used. See www.ncbi.nih.gov. One skilled in the art can readily determine the sequence of interest (wherein positions correspond to amino acids or nucleic acids in a reference sequence) by aligning the sequence of interest with a reference sequence using an appropriate BLAST program with default settings (e.g., for BLASTP: Gap open penalty: 11, Gap extension penalty: 1, Expectation: 10, Word length: 3, Maximum score: 25, Maximum alignments: 15, and Matrix: blosum62; and for BLASTN: Gap open penalty: 5, Gap extension penalty: 2, Nucleotide matches: 1, Nucleotide mismatches -3, Expectation: 10, Word size: 11, Maximum score: 25, and Maximum alignments: 15).

[0066] Preferred host cells are plant cells. In this article, recombinant host cells are genetically modified to contain isolated nucleic acid molecules, contain one or more deleted genes that are normally present and functional in host cells or are otherwise non-functional genes, or contain one or more genes that produce at least one recombinant protein. Nucleic acids encoding proteins of the present disclosure can be introduced by any method known in the art that is suitable for a particular type of cell (including but not limited to transformation, lipofection, electroporation, or any other method known to those skilled in the art).

[0067] "Isolated", "isolated DNA molecules" or equivalent terms or phrases are intended to indicate that a DNA molecule or other portion is present alone or in combination with other components, but is different from or not in its natural environment. For example, nucleic acid elements (such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc.) naturally present in the DNA of the genome of an organism are not considered "isolated" as long as the element is located in the genome of the organism and is located at the position within the genome where it is naturally found. However, as long as the element is not in the genome of the organism where it is naturally found, the element is different from its natural form, or the element is not located at the position within the genome where it is naturally found, each of these elements and sub-portions of these elements will be "isolated" from their natural environment within the scope of the present disclosure. Similarly, as long as the nucleotide sequence encoding a protein or any naturally occurring variant of the protein is not in the DNA of the organism where the sequence encoding the protein is naturally found in its natural location, or the nucleotide sequence is different from its natural form, the nucleotide sequence will be an isolated nucleotide sequence. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a cell of a plant, algae, fungus or bacteria, or a nucleotide sequence of DNA present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar construct used to transform cells, in the genome of a plant or bacteria, or in detectable amounts in tissues, progeny, biological samples or commercial products derived from a plant or bacteria.

[0068] Having generally described the compositions, methods and processes of the present disclosure, the same will be better understood by reference to certain specific examples which are included herein to further illustrate the disclosure and are not intended to limit the scope of the invention which is defined by the claims. Example

[0069] The present disclosure is further described in detail in the following examples, which do not limit the scope of the present disclosure in any way. The accompanying drawings are intended to be considered as an integral part of the specification and description of the present disclosure. The following examples are used to illustrate, but not to limit the content of the claimed protection. Example 1: Editing the rice photosystem II subunit S (OsPsbS1) gene using CRISPR / Cas9

[0070] This example describes a high-throughput process for screening novel alleles generated by CRISPR / Cas9 noncoding sequence mutagenesis upstream of OsPsbS1. The findings presented in this example reveal the ability to generate a wide range of phenotypes, including overexpression, with significant effects on NPQ and iWUE. Finally, the results identify a scheme of cis-regulation across allele libraries that may inform gene editing of overexpression within other features. Materials and methods Plant growth conditions

[0071] Rice cultivar Nipponbare (Oryza sativa ssp. japonica) seeds were placed on Whatman filter paper at 100 μmol m -2 s -1 Germination was carried out for seven days under fluorescent lights with a daily photoperiod of 14 hours day length (day temperature 27°C / night temperature 25°C). Seedlings were transplanted into soil including equal parts of Turface and Sunshine Mix #4 (Sungro) and grown under seasonal daylight (10-14 hours) in a south-facing greenhouse with fluctuating temperatures (high temperature 38°C / low temperature 16°C) and relative humidity (45-60%). Plants were fertilized with 0.1% Sprint 330 iron supplement after transplanting 2 weeks after germination and at the beginning of grain filling 10 weeks after germination. In addition, plants were fertilized monthly with JR Peter's Blue 20-20-20 fertilizer. Petri dishes were kept full of water to simulate flooded growing conditions. Genotypes were randomly distributed in the petri dishes and throughout the greenhouse to minimize location effects. At the V4-5 leaf stage, T 1 The progeny and WT controls were analyzed for differences in NPQ ability and sensitivity to the selectable marker hygromycin.

[0072] To evaluate differences in photosynthetic efficiency and yield, the gene-edited T 2 Plants were grown in a larger greenhouse with more uniform light exposure, field-related growth conditions (high 40°C / low 27°C, 30-50% relative humidity), without supplemental lighting. Plants were grown and yield data were collected, which coincided with the California rice growing season (April-October), after which the plants were dried for 2 weeks. Aboveground dry biomass, yield, and 1000 seed weight were quantified by destructive harvest. Construct cloning and guide RNA design

[0073] Eight guide RNA (gRNA) target sites were identified upstream of the rice functional PsbS ortholog OsPsbS1 (LOC_Os01g64960) using CRISPR-P (crispr[dot]hzau[dot]edu[dot]cn), and a 1.5 kb region upstream of the start codon of OsPsbS1 was identified in the draft genome of the indica rice subspecies (Oryza sativa ssp. indica) variety IR64 (Schatz et al., 2014, schatzlab[dot]cshl[dot]edu / data / rice / ). Then as previously described (Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci. 2015; 112 (11): 3570-3575. doi: 10.1073 / pnas.1420294112), 8 gRNA spacer sequences were assembled into DNA cassettes interspersed with scaffolds and tRNA linkers for multicistronic gRNA expression and synthesized (Genscript). The insert was cloned into the pRGEB32 rice Agrobacterium-mediated transformation vector (Addgene Plasmid # 63142) by GoldenGate Assembly to generate the pRGEB32_OsPsbS1_8xgRNA vector. In this vector, the OsU3 promoter drives the expression of a polycistronic gRNA-tRNA cassette with a scaffold, the ZmUbi promoter drives the expression of a binuclear localized SpCas9 optimized for rice codons, and the hygromycin resistance gene (HygR) is used for Agrobacterium-mediated transformation of embryogenic rice callus. The sequences are provided in Table 1 below. Table 1: OsPsbS1 non-coding sequence gRNA spacer sequence and position relative to the start codon In vitro gRNA validation of upstream OsPsbS1 noncoding sequences

[0074] Each candidate gRNA spacer sequence was synthesized as a ssDNA oligomer with additional 5' and 3' overhangs necessary for T7 RNA polymerase transcription and binding to the gRNA scaffold primer, respectively (Table 2). TMOligos were synthesized into dsDNA by high-fidelity PCR (NEB) and transcribed into RNA using the HiScribe RNA Synthesis Kit (NEB). The residual dsDNA was digested by DNAseI and purified using the RNeasy Mini Kit (Qiagen). Cas9 and each gRNA spacer sequence were incubated in 2 mM HEPES, 10 mM NaCl, 0.5 mM MgCl 2 , 10 μ m EDTA (pH 6.5) buffer at 37°C for 20 min, and then the complexed ribonucleoprotein was incubated with 100 ng of PCR-amplified 2 kb upstream region of OsPsbS1 (ssp. indica) at 37°C overnight to verify the activity of all 8 guide RNAs (Error! Reference source not found). Table 2: Oligos used for in vitro Cas9-gRNA editing of OsPsbs1 noncoding sequences in O. sativa (ssp. indica) Induction of embryonic callus

[0075] Mature seeds of rice (Oryza sativa ssp. japonica cv. Nipponbare) were hulled and surface sterilized for 20 minutes in 20% (v / v) commercial bleach (5.25% sodium hypochlorite) plus a drop of Tween 20. Three washes with sterile water were used to remove residual bleach from the seeds. The dehulled seeds were placed on callus induction medium (CIM) (N6 salts and vitamins (Chu, CC et al., Establishment of an efficient medium for anther culture of rice, through comparative experiments on the nitrogen sources. Sci Sin 18, (1975)), 30 g / L maltose, 0.1 g / L inositol, 0.3 g / L casein hydrolysate, 0.5 g / L L-proline, 0.5 g / L L-glutamine, 2.5 mg / L 2,4-D, 0.2 mg / L BAP, 5 mM CuSO 4 , 3.5 g / L Phytagel, pH 5.8) and incubated at 28°C in the dark to initiate callus induction. 6 to 8 week old embryogenic calli were used as targets for transformation. Agrobacterium-mediated transformation

[0076] The embryogenic callus was dried for 30 minutes and then incubated with a suspension of Agrobacterium tumefaciens EHA105 carrying the cloned binary vector pRGEB32_OsPsbS1_8xgRNA (OD 600nm =0.1). After 30 minutes of incubation, the Agrobacterium suspension was removed. The callus was then placed on a sterile filter paper and transferred to a co-culture medium (N6 salts and vitamins, 30 g / L maltose, 10 g / L glucose, 0.1 g / L inositol, 0.3 g / L casein hydrolysate, 0.5 g / L L-proline, 0.5 g / L L-glutamine, 2 mg / L 2,4-D, 0.5 mg / L thiamine, 100 mM acetosyringone, 3.5 g / L phytagel, pH 5.2) and incubated at 21 ° C in the dark for 3 days. After co-cultivation, callus was transferred to resting medium (N6 salts and vitamins, 30 g / L maltose, 0.1 g / L inositol, 0.3 g / L casein hydrolysate, 0.5 g / L L-proline, 0.5 g / L L-glutamine, 2 mg / L 2,4-D, 0.5 mg / L thiamine, 100 mg / L timentin, 3.5 g / L phytagel, pH 5.8) and incubated at 28°C in the dark for 7 days. Callus was then transferred to selection medium (CIM plus 250 mg / L cefotaxime and 50 mg / L hygromycin B) and allowed to proliferate at 28°C in the dark for 14 days. Well-proliferated tissue was transferred to CIM containing 75 mg / L hygromycin B.

[0077] The remaining tissues were subcultured on fresh selection medium every 3 to 4 weeks. When a sufficient amount (about 1.5 cm in diameter) of the putative transformed tissues were obtained, they were transferred to a regeneration medium (MS salts and vitamins (Murashige, T. & Skoog, FA Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15, (1962)) containing 40 mg / L hygromycin B, 30 g / L sucrose, 30 g / L sorbitol, 0.5 mg / L NAA, 1 mg / L BAP, 150 mg / L cefotaxime) and grown at 26 ° C, 16 hours of light, 90 μmol photons m - 2 s -1When the regenerated plantlets grew to at least 1 cm in height, they were transferred to rooting medium (MS salts and vitamins, 20 g / L sucrose, 1 g / L inositol, 150 mg / L cefotaxime) containing 20 mg / L hygromycin B and incubated at 26 °C with 16 h of light (150 μmol photons m -2 s -1 ) and dark for 8 hours until roots are formed and leaves touch the phytatray lid. When possible, multiple regenerates are recovered per callus. Plantlets are then transferred to soil. Chlorophyll fluorescence measurement of NPQ

[0078] Leaf punches were sampled from mature, fully developed leaves at the V3-5 leaf stage and floated in 270 μL of water in a 96-well plate. Plates were adapted in the dark for at least 30 min prior to analysis. In vivo chlorophyll fluorescence measurements were determined at room temperature using an Image-PAM Maxi (Walz) pulse amplitude modulated fluorimeter. After dark adaptation (F 0 、F m ) and light adaptation period (F 0 '、F m The fluorescence level of ') was monitored in two ways.

[0079] To resolve phenotypically segregating OsPsbS1 NCS gene-edited alleles from a single parent, individual leaf punches at the V3 leaf stage were exposed to high-intensity actinic light (1500 μmol m -2 s -1 ). Putative homozygous lines were identified as those progeny in the top and bottom 10% of total NPQ. Leaf punches were then used to determine hygromycin sensitivity and Cas9 transgene segregation as described.

[0080] Candidates identified as lacking Cas9 were resampled at the V5 leaf stage and two leaf punches from two mature leaves per plant were phenotyped to compare NPQ relative to Nipponbare WT plants. High-intensity actinic light (1500 μmol m -2 s -1 ) and 10 min dark relaxation (0 μmol m -2 s -1 ) Quantitative NPQ. The NPQ in both cases was calculated using the following formula. NPQ = [(F m -F m ') / F m '] (1) Hygromycin sensitivity assay for high-throughput Cas9 transgene detection

[0081] Floating leaf punches for NPQ capacity were then used to determine hygromycin sensitivity and segregation of the transgene. Hygromycin B (50 mg / mL 1× PBS) was added to each well to a final concentration of 20 μg / mL of the antibiotic. The plates were incubated for three days under rice germination chamber conditions and then imaged using the Image-PAM Maxi (Walz) to identify photosystem II (F v / F m ) is calculated using the following formula. F v / F m = (F m – F o ) / (F m ) (2) Sensitive, non-GM plants F v / F m Typically decreased by >0.3-0.4, while transgenic plants maintained WT F of ∼0.7-0.8 v / F m . Total RNA / protein extraction from representative lines with different NPQs

[0082] At noon, 2 cm leaf tissue was collected from the youngest, fully developed leaves and cultured in a lysing medium containing FastPrep-24 TM ) in RNase-free and DNase-free tubes. TM Leaf tissues were ground on dry ice using a high-speed homogenizer (6.0 m / s for 2×40 s, MP Biomedical). Protein and mRNA were extracted from the same leaf samples (NucleoSpin RNA / Protein Kit, REF740933, Macherey-Nagel GmbH & Co., Düren, Germany). Quantitative RT-PCR of OsPsbS1 relative to two reference genes

[0083] The extracted mRNA was treated with DNase (ThermoFisher Scientific) and then converted to cDNA using Omniscript reverse transcriptase (Qiagen) and a 1:1 mixture of random hexamers and oligo dT according to the manufacturer's recommendations. Quantitative reverse transcription PCR was used to quantify OsPsbS1 transcripts relative to OsUBQ and OsUBQ5 transcripts, and qRT-PCR expression was normalized to multiple reference genes in three biological replicates with technical replicates using published methods (Vandesompele, J. et al., Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, (2002); Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8, R19 (2007)). Samples were run on a 7500 Fast Real-Time PCR System (Applied Biosystems, Ghent, Belgium) with a total volume of 20 μL using 4 μL of 1:10 diluted cDNA. Primers were empirically validated on a 5-step dilution series (1:1 to 1:81) of WT cDNA. All final primer pairs had amplification efficiencies between 90-105% and linear amplification over the dynamic range tested. A single peak was observed for each gene of interest in the melt-curve analysis, validating the specificity of the amplicon. UBQ and PsbS1 were described in Fu, X et al. OsbZIP72 and OsMYBS2 regulate transcription of OsPsbS1 in rice in an opposing cooperation.New Phytol 229, 370–387 (2021), and UBQ5 was described in Jain, M., Nijhawan, A., Tyagi, AK & Khurana, JP Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochemical and Biophysical Research Communications 345, 646–651 (2006). In the morning (. Fig. 9A ),night( Fig. 9B ) and the time course of plants grown in growth chambers ( Fig. 9C ) measured transcription levels in greenhouse grown plants. Primers and primer efficiencies are shown in Table 3 below. Table 3: qRT-PCR primer pairs and observed primer efficiencies in WT across a 1:81 dilution series Immunoblotting of whole leaf protein extracts

[0084] The precipitated proteins were resuspended in the supplied protein solubilization buffer (PSB-TCEP) and quantified using TCA colorimetric analysis (Karlsson, JO, Ostwald, K., Kabjorn, C. & Andersson, MA Method for Protein Assay in Laemmli Buffer. Analytical Biochemistry 219, 144–146 (1994)). TMGel (BIO-RAD) analysis containing 5 μg of total protein samples, via wet transfer method transferred to polyvinylidene difluoride membrane (Immobilon-FL 0.45 μm, Millipore), and blocked with 3% skim milk powder for immunodetection. Cut the membrane and incubate with the following antibodies. Rabbit polyclonal antibodies cultivated for sorghum PsbS (SbPsbS) were generously shared by Steven J.Burgess (University of Illinois) and used at a dilution of 1:2,500. Rabbit polyclonal antibodies cultivated for synthetic peptides of the β subunit (Atpβ) of ATP synthase were obtained from Agrisera (Catalog No. AS05085) and used at a dilution of 1:10,000. After incubation with HRP-coupled anti-rabbit secondary antibodies (1:10,000 dilution) from GE Healthcare, SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific) was used to detect bands by chemiluminescence. Protein bands were quantified by densitometry using ImageQuant TL software (version 7.0 GE Healthcare Life Sciences, Pittsburgh, PA, USA). PsbS abundance was quantified relative to WT based on a dilution series of WT PsbS protein. Steady-state NPQ adapted to low, medium, and high light

[0085] To assess steady-state differences in NPQ capacity across representative lines, genotypes differing in PsbS abundance were grown and phenotyped in the youngest fully expanded leaves of 8-week-old vegetative rice plants. Whole plants were dark-adapted for 1 h and phenotyped according to the following protocol, with quantification of chlorophyll fluorescence every 3 min by a multiphase flash program (LI6800, LI-COR, Lincoln, NE, USA): 0 μmol m for 15 min and 10 μmol m for 25 min. -2 s -1 –45 min 100 μmol m -2 s -1 –45 min 800 μmol m -2 s -1 – 45 min 1500 μmol m -2 s -1 –15 min 0 μmol m -2 s -1 , using increasing red light intensity and a total of 10 μmol m -2 s -1 Blue light was used for all light-dependent measurements. In addition to Equations 1 and 2 (above), the operating efficiency of PSII (ΦPSII) was monitored using the following equation: ΦPSII=(Fm '-F s ') / F m ' (3) Parallel gas exchange and chlorophyll fluorescence analysis under increased red light

[0086] Photosynthetic gas exchange kinetics were measured in the youngest, fully expanded flag leaves of 12-week-old flowering rice plants. 2 Gas exchange measurements were performed using a leaf chamber and an open-type gas exchange system with an integrated modulated fluorimeter (LI6800, LI-COR, Lincoln, NE, USA). The whole plant was adapted to low light for 1–2 h to alleviate the photosynthetic decline in the afternoon and to darkness for at least 30 min to allow for simultaneous monitoring of gas exchange (e.g., A n , g sw ) and chlorophyll fluorescence parameters (e.g. F v / F m For all measurements, the room conditions were set at 400 ppm room CO 2 , 27°C room temperature, 1.4 kPa leaf vapor pressure deficit, 500 μmol s -1 Flow rate and fan speed of 10,000 rpm. Samples were analyzed over the ambient day length range (8am–5pm).

[0087] Monitor steady-state photosynthesis and stomatal conductance in response to changes in red light intensity (100% red LED, λ 峰值 The light intensity was varied from 0, 50, 80, 110, 140, 170, 200, 300, 400, 600, 800, 1000, 1500 and 2000 μmol m -2 s -1 Each light step was adapted for 10-20 minutes. sw The slope is maintained at less than 0.005 + / - 0.00025 SD over a period of 40 seconds and when the net assimilation rate A n Steady state was reached when the change was less than 0.5 + / - 0.25 SD over a 20 s period. Net assimilation rate, stomatal conductance, and intracellular [CO 2 ]. Saturation pulses were then applied using a multiphase flash program to collect all relevant chlorophyll fluorescence parameters. In addition to NPQ and F v / F m In addition, the intrinsic water use efficiency (iWUE) (Equation 4, below), ΦPSII (Equation 2, above), and Q ARedox state (Eq. 5, below). Derivation of 1-qL assumes a "lake" model for the photosynthetic antenna complex. iWUE = A n / g sw (4) 1-qL=1- (F q ' / F v ') / (F o ' / F s ') (5) PCR genotyping of transgene-free edited lines

[0088] Bead beating (Lysing Matrix D, FastPrep-24 TM ) 50 mg of leaf tissue was ground and genomic DNA was extracted in 2×CTAB buffer at 65°C for 15 minutes. DNA was isolated via chloroform phase separation and precipitated with isopropanol. The precipitate was briefly washed in 70% ethanol, then dried and resuspended in 1×TE buffer.

[0089] Using 5×GC enrichment buffer, the TM High-fidelity PCR was used to amplify two overlapping regions spanning eight gRNA target sites (Table 4). PCR products were amplified from at least two putative homozygotes per line and sequenced by Sanger sequencing. Snapgene was used to assemble a ~4.3-kb contig of the upstream region. Table 4: Primers used for PCR amplification and genotyping of OSPSBS1 NCS Pacbio long-read whole-genome sequencing and analysis

[0090] Leaf tissue for high molecular weight genomic DNA (HMW gDNA) extraction was starved for 4 days in the dark, then sampled and snap frozen in liquid nitrogen. DNA was isolated using the NucleoBond HMW DNA Kit (TakaraBio, catalog #740160.2) with the following modifications: plant leaves were ground by pestle and mortar under liquid nitrogen, where 1 g of ground leaf tissue was resuspended in 2.5 times the amount of recommended lysis buffer and in a 50°C water bath for 4 hours. The amount of Binding Buffer H2 was also increased proportionally. HMW gDNA was resuspended in water by gently pipetting, and quality was assessed by Femto pulse analysis (median fragment length 13-15 kb).

[0091] HMW gDNA was pooled and sequenced by HiFi circular consensus sequencing (CCS) on a single 8M SMRT cell using PacBio Sequel II (QB3 Genomics, UC Berkeley, Berkeley, CA, RRID: SCR_022170). Uncurated reads from overexpression lines N2-4_OX and N19-1_OX were first quality checked using FastQC (www[dot]bioinformatics[dot]babraham[dot]ac[dot]uk / projects / fastqc / ). Reads were mapped to the rice (Oryza sativa) v7.0 reference genome (Ouyang, S. et al., The TIGR Rice Genome Annotation Resource: improvements and new features. Nucleic Acids Research 35, D883–D887 (2007)) using pbmm2 (a Pacific Biosciences wrapper for minimap2 specifically developed for HiFi reads) and assembled using pbIPA (Pacific Biosciences phased assembler (github[dot]com / PacificBiosciences / pbbioconda)). Sniffles (github[dot]com / fritzsedlazeck / Sniffles) and pbsv (github[dot]com / PacificBiosciences / pbbioconda) were used to identify structural variants from the mapped reads. D-Genies (D-GENIES: dot plot large genomes in an interactive, efficient and simple way [PeerJ]. Peerj [dot] com / articles / 4958 / ) was used to align the de novo assembly with the reference genome and generate dot plots. Integrative Genomics Viewer (Robinson, JT et al., Integrative genomics viewer. Nat Biotechnol 29, 24–26 (2011)) was used to visualize mapped reads, aligned assemblies, and sniffles structural variation outputs. result Independent event libraries upstream of OsPsbS1 for multiplexed editing

[0092] To generate novel cis-regulatory variants upstream of OsPsbS1 (LOC_Os01g64960), eight specific and evolutionarily conserved guide RNA (gRNA) target sites (Table 1) were introduced into rice (Oryza sativa ssp. japonica) cultivar Nipponbare callus via Agrobacterium-guided transformation. This design avoided targeting a putative QTL for NPQ activity in the upstream of rice OsPsbS1, which is a previously identified internal 2.7 kb japonica-specific insert (Kasajima, I. et al., Molecular distinction in genetic regulation of nonphotochemical quenching in rice. Proceedings of the National Academy of Sciences 108, 13835–13840 (2011); Wang Q, Zhao H, Jiang J et al., Genetic Architecture of Natural Variation in Rice Nonphotochemical Quenching Capacity Revealed by Genome-Wide Association Study. 2017; 8 (October). doi: 10.3389 / fpls.2017.01773) ( Figure 1A Twenty-three independent fertile transformants were generated ( Figure 1B ). When possible, multiple sister lines were recovered from the same transformed callus, resulting in 78 T 0 Clonal lines recovered from the same transformation event are distinguished by a hyphen, where the number before the hyphen corresponds to the event number and the number after the hyphen corresponds to the line number (e.g., for event #2, clonal lines would be designated 2-1, 2-2, 2-3, etc.). High-throughput chlorophyll fluorescence screening of edited, semi-dominant PsbS alleles

[0093] In vivo chlorophyll fluorescence screening was used to screen for 78 diploid T 0 The unique phenotypes of the transformants were analyzed, resulting in up to 156 gene-edited alleles. 0 The phenotype of the next generation can be somatic, and competition between alleles can mask the dynamic changes in gene expression and activity. To overcome this problem, in T 1Each transformant is screened in the next generation, where the allele is fixed to homozygosity and the hemizygous Cas9 transgene can be segregated out. Based on the Mendelian segregation of genetic alleles and a single insertion of the transgene, it is expected that 1 / 4 of the T 1 Offspring (by T 0 The lines were selfed) and were homozygous for each mutated cis-regulatory element, and 1 / 4 of the progeny were expected to have lost the transgene carrying Cas9 and the plant antibiotic resistance cassette ( Figure 2A ). Relying on the fact that CRISPR / Cas9 mainly produces biallelic mutations, 1 Progeny screening can help identify progeny that are heterozygous [A / a] or homozygous for either OsPsbS1 allele [A / A or a / a], and phenotypic differences between the two alleles (designated A and a).

[0094] PsbS is a gene in which loss-of-function mutations appear semidominant. These mutations show a strong linear correlation between copy number and NPQ capacity (r 2 =0.9235)( Figure 2B ). Nevertheless, T 0 The maximum NPQ is the average T 1 Poor predictors of goodness of fit (r 2 =0.2675). T 0 The phenotype overestimates the putative T 1 The number of overexpressors was not significantly increased, and no candidate stable overexpressors were identified ( Figure 2C ). In order to resolve stable and heritable alleles, T 1 Progeny populations were screened at high throughput to determine differences in NPQ competence and Cas9 transgene segregation. Putative homozygous alleles (A / A and a / a, Figure 2D ), where more subtle differences in population phenotypes were resolved by grouping the upper and lower 10% of the NPQ phenotype. Leaf punches for evaluation of NPQ capacity were treated with the plant-selective antibiotic hygromycin and the quantum efficiency of photosystem II (F v / F m ), a common indicator of plant stress and light damage ( Figure 2E-2F ). Significant differences were observed after only 72 h of incubation, with F v / F m Using this method, homozygous, Cas9-null plants can be identified after <2 h of work, allowing for rapid identification of fixed accessions for entry into T 2 generation. Gene-edited overexpression alleles exist, albeit rarely

[0095] By comparing WT plants and plants from a single T 0 Pairwise comparisons between progeny of the parents identified putative homozygous alleles, as depicted ( Figure 2D To assess phenotypic variation across all putative stable alleles, the maximum NPQ of all 120 phenotypically resolved alleles was plotted ( Figure 3A ).

[0096] Nearly two-thirds of the 120 stable alleles isolated had NPQ capabilities similar to WT (61.67%), with the second and third largest groups being knockout phenotypes (21.67%) and knockdown phenotypes (15%), respectively. Two independent overexpression alleles were isolated, comprising 1.67% of the total phenotypic variance ( Figure 3B ). Chlorophyll fluorescence phenotype is correlated with PsbS protein abundance

[0097] To better understand the resulting phenotypic variation, representative lines from the two events that generated the overexpression allele (i.e., event 2 and event 19) were evaluated for their ability to adapt to low, medium, and high light conditions ( Figure 4A ). The detected individuals are in the initial F v / F m ( Figure 4B ) or NPQ at 100 μmol m-2s-1 ( Figure 4C ) showed no differences. At 800 μmol m-2s-1 of light, the event 2-5 knockout strain (2-5_KO) and the event 2-1 strong knockout (2-1_SKD) strain showed significantly reduced NPQ capacity relative to the event 2WT control (p<0.0001 and p=0.0041, respectively). In contrast, the event 19-1 overexpression strain (19-1_OX) showed significantly higher NPQ capacity than the event 19WT control under the same conditions (p=0.0001). At 1500 μmol m-2s-1, significant differences were observed between all non-WT-like alleles (p<0.0003) ( Figure 4C ).

[0098] Immunoblot analysis of OsPsbS1 protein highlighted the absolute protein abundance ( Figure 4D ) and relative to the abundance of the Nipponbare WT control dilution series ( Figures 4E-4F) dynamic range. In event 2, the 2-1_SKD allele and the 2-6 weak knockdown allele (2-6_WKD) accumulated 1 / 8 and 1 / 4 of the WT OsPsbS1 protein level, respectively. The 2-4 overexpression allele (2-4_OX) accumulated 2-4-fold of the WT OsPsbS1 protein, similar to 19-1_OX. 2-5_KO was below the threshold for PsbS detection. It was possible to fit all genotypes at 1500 μmol m when normalizing to the loading control for each membrane, chloroplast Atpβ, and the Nipponbare WT control to allow comparison across experiments. -2 s -1 The logarithmic relationship between OsPsbS1 protein abundance and NPQ capacity (r 2 =0.7872, which is consistent with the previous study in Arabidopsis thaliana 119 ) is consistent with the PsbS dose-dependent activity observed in ( Figure 4G ).

[0099] NPQ is photoprotective but can compete with light harvesting. To assess the extent to which photosynthesis is limited in the isolated overexpression alleles, ΦPSII (the efficiency of PSII operating under light) was plotted relative to NPQ for WT and OX lines under all three light conditions ( Figure 4H A strong negative correlation was observed (r 2 =0.9684), among which the individual with the highest NPQ (i.e., 19-1_OX) had a -2 s -1 and 1500 μmol m -2 s -1 To determine the extent to which NPQ is photoprotective, the F was measured 15 min after the last illumination step of the assayed illumination regimen. v / F m Relative to 2-4_OX, 2-5_KO, 2-1_SKD, and 2-6_WKD alleles showed higher amounts of photoinhibition, as shown in F v / F m The decrease indicated by (p<0.0001, p=0.011, p=0.024, respectively) ( Fig. 4I ). For each event, there was no phenotype difference between the overexpression and WT-like alleles. However, for the last 1500 μmol m -2 s -1 The NPQ capability of the lighting scheme after F v / F m Plotting reveals a strong correlation between NPQ capacity and the quantum efficiency of maintaining PSII ( Figure 4J). Therefore, the homeostatic adaptation of rice lines under different PsbS abundances and light intensities revealed a possible trade-off between light harvesting and photoprotection at different PsbS levels. Differences in PSBs abundance alter the steady-state gas exchange phenotype in response to red light

[0100] Previous studies have shown that transgenic overexpression of PsbS does not impair the steady-state CO2 in rice. 2 Assimilation (Hubbart, S. et al., Enhanced thylakoid photoprotection can increase yield and canopy radiation use efficiency in rice. Commun Biol 1, 22 (2018); Hubbart, S., Ajigboye, OO, Horton, P. & Murchie, EH The photoprotective protein PsbS exertscontrol over CO 2 assimilation rate in fluctuating light in rice: Photoprotection and CO2 assimilation in rice. The Plant Journal (2012) doi: 10.1111 / j.1365-313X.2012.04995.x), but in tobacco it did increase intrinsic water use efficiency (iWUE) under red light ( K. et al., Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop. Nat Commun 9, 868 (2018). To determine the extent to which these phenotypes are consistent across rice genotypes with varying endogenous PsbS abundance, steady-state chlorophyll fluorescence and gas exchange parameters were assessed in six genotypes under increased red light ( Figures 5A-5E ).

[0101] High-throughput screening with NPQ ( Figure 3A-3B ) and adapt to low light, medium light, and high light intensities ( Figures 4A-4J ) were consistent with the knockdown lines 2-1_SKD and 2-6_WKD at >500 μmol m -2 s -1showed significantly lower NPQ (p<0.0001) relative to the Nipponbare WT control at light intensities of. While 2-4_OX showed significantly higher NPQ (p<0.0001) only at 1500 and 2000 μmol m -2 s -1 . The 2-4_WT sample line showed a moderate decrease in NPQ (0.002 < p < 0.0004) at 500 μmol m Figure 5A . While the second gene-edited WT-like control (2-7_WT-like) showed no significant differences from the Nipponbare WT across all measured phenotypes ( -2 s -1 and 1500 μmol m -2 s -1 ( Figures 5A-5E ). The mild knockdown of 2-6_WKD increased ΦPSII (0.041 < p < 0.006) at light intensities above 500 μmol m -2 s -1 ( Figure 5B ).

[0102] Consistent with the increased ΦPSII, the weak knockdown of PsbS also increased steady-state CO -2 s -1 assimilation at light intensities above 500 μmol m 2 with a statistically significant increase (p = 0.016; p < 0.001) ( Figure 5C ). Stomatal conductance was also significantly higher in the 2-6_WKD line compared to the Nipponbare WT (p = 0.043; p = 0.0007), with a more moderate increase in stomatal conductance observed in the 2-1_SKD line (p = 0.043 - 0.048) ( Figure 5D ).

[0103] To evaluate the differences in iWUE due to changes in PsbS activity, the iWUE for each replicate at high light (>600 μmol m -2 s -1 ) was averaged and plotted against the maximum NPQ. A significant positive correlation was observed between the increased iWUE and the higher maximum NPQ capacity (p = 0.019 for all data points, p = 0.007 for the mean) ( Figure 5E ). Q A redox state was an inconsistent predictor of iWUE in rice g sw . Predictions that the increased iWUE phenotype observed in transgenic tobacco was mediated by the Q

[0104] redox state, where the QA redox state is approximated by 1 - qL ( A ) K. et al., Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop. Nat Commun 9, 868 (2018)). Surprisingly, there was no significant difference in 1-qL across lines with different OsPsbS compared to the Nipponbare WT control ( Fig. 6A ).

[0105] To further explore the putative correlations, 1-qL was plotted against stomatal conductance for all genotypes and light intensities. Although significant nonlinearity (p < 0.0001) and relatively strong goodness of fit (r 2 =0.634, average value r 2 =0.846)( Figure 6B ), but this correlation was stronger than in tobacco (r 2 =0.98, p<0.001) reported a weaker ( K. et al., Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop. Nat Commun 9, 868 (2018). With the greater dynamic range of NPQ capabilities revealed in this study, correlations were assessed individually across all six tested genotypes ( Figure 6C ). Although the goodness of fit has improved significantly (r 2 >0.974), but the linear regression slopes between Nipponbare WT and 2-4_OX were significantly different (p=0.007), and the y-intercepts of the other non-WT lines were significantly different (p<0.001) ( Fig.6D ). In summary, except for the 2-7_WT sample control, all Q A The linear regressions for both redox state and stomatal conductance were significantly different from Nipponbare WT due to the different PsbS levels. Most non-WT phenotypes are explained by variations in the 5'UTR

[0106] Differences in NPQ capacity, protein abundance, and iWUE confirmed that the observed phenotypic variation was due to noncoding sequence mutagenesis. To assess the underlying mutational cause of these phenotypes, the ∼4.3-kb upstream cis-regulatory region was sequenced by Sanger sequencing. 107 of the 120 unique alleles ( Figures 7A-7C ) could be amplified by PCR (89.2%).

[0107] Three alleles were observed that contained large deletions of five distal gRNA target sites ( Fig. 7A Interestingly, NPQ of all three lines was indistinguishable from WT ( Figure 7B ). Therefore, it is likely that the majority of the observed phenotypic variation can be explained by proximal gRNA variants within and near the 5'UTR. Cis-regulatory analysis of event 2 lines with different NPQs ( Figures 4A-6D ) revealed distinct 5'UTR deletions. The size and relative position of the deletions corresponded to knockout (KO) and knockdown (KD) phenotypes, with the majority of variants driven by the second proximal gRNA ( Figure 7C As shown in the 24-5_18WT-like allele, the transcription start site (TSS) is not essential for OsPsbS1 expression. Long-read sequencing resolves complex structural variants underlying overexpression

[0108] Unexpectedly, none of the overexpressed alleles (event 2-4OX or event 19-1_OX) could be analyzed by PCR genotyping. To determine if the underlying cause of these phenotypes was complex structural variants, homozygous 2-4_OX T was sequenced by long-read HiFi circular consensus sequencing (Pacific Biosciences). 2 Strains and isolates 19-1_OX T 1 Due to unexpected sterility, the T 1 The 19-1 allele was sequenced in the first generation and the sterility was also found in 5 of the first 28 independent transformants ( Figure 1B ). Reads were assembled de novo and mapped to the Nipponbare rice (O. sativa) v7.0 reference genome using bioinformatics tools developed by Pacific Biosciences (Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8, R19 (2007)) (see Materials and Methods, Error! Reference source not found).

[0109] First, all sequences and structural variants across 12 rice chromosomes were visualized by dot plots. Specifically, the dot plots of 2-4_OX and 19-1_OX were generated, and the OX variants (x-axis) of order-checking were plotted (data not shown) for reference genome (y-axis). It was found that the OX sequence was identical to the reference genome to a great extent. It was found that little chromosome variants (e.g., insertion and deletion, SNP) had a highly consistent pattern between 2-4_OX and 19-1_OX, which may represent the inherent sequence differences between the reference genome and the laboratory strain used. The two OX strains of order-checking really show moderate differences on the chromosome level structure, which is represented by the gap indicating larger insertion and deletion in continuity.

[0110] Improved resolution of the chromosome 1 locus of OsPsbS1 highlights large differences in structural variation that cannot be observed on a genome-wide scale. Fig. 8A In the figure, dot plots of increasing resolution of the chromosome 1 locus (1.1 Mbp) containing OsPsbS1 in the 2-4_OX line are shown, with breaks in continuity indicating the presence of genomic inversions. This was further confirmed at the sequence level by visualization using the Integrative Genome Viewer (IGV). Figure 8B The 2-4_OX line shows an increase in resolution of the chromosome 1 locus with an inversion of ∼254 kb upstream of OsPsbS1 (Chr1:37693233–37948089). The 19-1_OX line, however, showed no significant difference in the increase in resolution of the chromosome 1 locus (∼1.5 Mbp) containing OsPsbS1 ( Figure 8C However, increased resolution of the chromosome 1 locus in the 19-1_OX line revealed the presence of a ~3-4 kb inversion upstream of OsPsbS1 (Chr1: ~37693800–37696800)( Fig.8D ). The exact junction of the inversion was not resolvable by long-read sequencing but corresponds to the region between gRNA5 and gRNA7 listed in Table 1. Thus, inversions of different sizes explain the observed PsbS overexpression phenotype. The overexpression phenotype is constitutive, but expression is not

[0111] exist Figures 9A-9B In Figure 2, the expression levels of OsPsbS1 in samples collected at one time point are shown. Fig. 9A In the results, it can be seen that the expression of OsPsbS1 in the 2-4_OX strain was significantly higher than that in the wild type (p<0.01), and the transcription level of OsPsbS1 in the 2-5_KO strain was significantly lower and below the detection threshold. Fig. 9BAmong them, the expression levels in the 2-4_OX, WT, and 2-4_WT sample lines were comparable to each other, while the expression levels in the 2-6_WKD, 2-1_SKD, and 2-5_KO lines were lower than those in the WT, at a statistically significant level (0.01 < p < 0.0001). To clarify the observed differences, a growth chamber time course was completed for the 2-4_OX and WT plants. Fig. 9C The results showing this time course revealed that the expression in the OX line peaked in the morning and decreased over the course of the day, with a statistically significant difference in expression observed only at the first time point (1 hour, p < 0.01). Diurnal NPQ measurements showed that, compared to the WT, the NPQ levels in the 2-4_OX line remained high over the course of the day, unaffected by the changes seen in the transcriptional level of OsPsbS1. These results unexpectedly showed that the overexpression phenotype (i.e., NPQ) was constitutive, but the overexpression of OsPsbS1 was not. Discussion

[0112] As the understanding of basic biology deepens, the demand for specific and efficient gene editing tools to improve the application of biology in agriculture and medicine is also growing. If the design principles regulating gene expression are fully understood, the CRISPR / Cas9 toolkit has the potential to meet this demand. The results shown in this example demonstrate the ability to use CRISPR / Cas9 to mutagenize non-coding sequences, thus achieving natural gene overexpression at levels competitive with transgenic overexpression. These results benefited from a high-throughput screening process for rapidly detecting homozygous, Cas9-free progeny, which revealed a variety of array phenotypes.

[0113] Consistent with a large body of literature, it was found that the overexpression of PsbS increased the NPQ capacity (Li, X.-P., Müller-Moulé, P., Gilmore, A.M. & Niyogi, K.K. PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proceedings of the National Academy of Sciences 99, 15222–15227 (2002); Hubbart, S. et al., Enhanced thylakoid photoprotection can increase yield and canopy radiation use efficiency in rice. Commun Biol 1, 22 (2018); K. et al., Photosystem IISubunit S overexpression increases the efficiency of water use in a field-grown crop. Nat Commun 9,868 (2018)).

[0114] While the observed phenotypes reinforce the observed differences in agronomic traits that depend on PsbS activity, the most important takeaways from these results come from structural variants that cause fundamental changes in gene expression. Figure 1A ), we found that the distal region of the OsPsbS1 promoter, which is conserved between indica and japonica rice subspecies, is not essential for the expression of OsPsbS1 ( Figure 7B ), and most of the observed phenotypic variation is due to indels near and within the 5'UTR.

[0115] Surprisingly, the two overexpressed alleles that increased NPQ capacity were driven by inversions upstream of OsPsbS1 ( Fig. 8A -8F). Allele 19-1_OX provides an interesting example because it carries a clear inversion between the distal and proximal gRNAs, which can be easily replicated or introduced into other variants.

[0116] Analysis of long-read sequencing reveals the prevalence of structural variants in the genome. Complex structural variants (CSV), including translocations, insertions, and inversions, persist at both the population and genome-wide levels, such as in tomato (Alonge, M. et al., Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato. Cell 182, 145-161. e23 (2020)), rapeseed (Song, J.-M. et al., Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Brassica napus. Nat. Plants 6, 34–45 (2020)), maize (Sun, S. et al., Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes. Nat Genet 50, 1289–1295 (2018)), grapevine (Zhou, Y. et al., The population genetics of structural variants in grapevine domestication. Nat. Plants 5, 965–979 (2019)) and rice (Qin, P. et al., Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations. Cell 184, 3542-3558.e16 (2021)), affecting more than 20% of all genes.In fact, much of the current understanding of structural variants is driven by the prevalence of these changes that drive various human cancers and diseases (Weischenfeldt, J., Symmons, O., Spitz, F. & Korbel, J. O. Phenotypic impact of genomic structural variation: insights from and for human disease. Nat Rev Genet 14, 125–138 (2013); Beyter, D. et al., Long-read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits. Nat Genet 53, 779–786 (2021); Hehir-Kwa, J. Y. et al., A high-quality human reference panel reveals the complexity and distribution of genomic structural variants. Nat Commun 7, 12989 (2016)). However, unlike humans, plants exhibit greater tolerance to (and therefore abundance of) structural variants, which may be driven in vivo by transposable elements and recombination—both of which have been documented as key drivers of crop domestication (Domínguez, M. et al., The impact of transposable elements on tomato diversity. Nat Commun 11, 4058 (2020); Gaut, BS, Seymour, DK, Liu, Q. & Zhou, Y. Demography and its effects on genomic variation in crop domestication. Nature Plants 4, 512–520 (2018)).

[0117] In fact, chromosomal inversions have also been implicated in gene overexpression during peach domestication (Zhou, H. et al., A1.7-Mb chromosomal inversion downstream of a PpOFP1 gene is responsible for flat fruit shape in peach. Plant Biotechnology Journal 19, 192–205 (2021)), and complex rearrangements that are the cause of multiple myeloma (Affer, M. et al., Promiscuous MYC locusrearrangements hijack enhancers but mostly super-enhancers to dysregulate MYCexpression in multiple myeloma. Leukemia 28, 1725–1735 (2014)). Recently, Lu et al. showed that CRISPR / Cas9 can be used to drive natural overexpression via promoter exchange (Lu, Y. et al., A donor-DNA-free CRISPR / Cas-based approach to gene knock-up in rice. Nat. Plants 7, 1445–1452 (2021)), which generated inversions that increased OsPPO1 gene expression at different frequencies in ∼3% of transformed callus. However, these inversions came at the expense of expression of the opposite promoter, thereby knocking out gene expression of the target Calvin-Benson cycle protein 12 (OsCP12) gene (LOC_Os01g19740). In the case of the 19-1_OX allele, another inversion breakpoint occurred downstream of the 3'UTR of a neighboring gene (LOC_Os01g64930) and may not interfere with its expression. The work presented here (due to unbiased targeting design) strengthens and expands the potential of inversions for genome engineering of natural gene overexpression.

[0118] In the dataset, overexpressed alleles containing inversions accounted for 2 of the 13 unique alleles that could not be PCR amplified and sequenced. Of the remaining 11 alleles, 8 were phenotypic knockouts, 1 was phenotypic knockdown, and 2 had NPQ activity similar to WT. CSV also underlies these remaining alleles, and it is possible to increase the potential frequency of CSV alleles from 1.7% to 10.8% by CRISPR / Cas9. Example 2: Promoter and 5'UTR mutagenesis of OsVDE / OsZEP in Nipponbare 2-4OX PsbS strain

[0119] This example describes targeting rice (O. sativa) VDE (OsVDE, LOC_Os04g31040) and ZEP (OsZEP, LOC_Os04g37619) via CRISPR / Cas9 mutagenesis to reproduce the VPZ phenotype via altered endogenous gene expression (Kromdijk J, Glowacka K, Leonelli L et al., Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science. 2016; 354(6314): 857-862). The PsbS OX line described in Example 1 was used as a recipient parent, and OsVDE and OsZEP were individually targeted in separate transformant pools to identify overexpressed alleles in each trait. Candidate OX and knockdown (KD) alleles were crossed and evaluated for faster relaxation of NPQ. Materials and Methods Guide RNA Design and Construct Cloning

[0120] Five to eight guide RNA (gRNA) target sites were identified within the promoter and 5'UTR upstream of OsVDE and OsZEP. The promoter is broadly defined as the 2kb region upstream of the start codon (ATG) of the gene of interest, but in the case of OsVDE, the upstream gene limits the putative promoter and 5'UTR to ~750bp. Candidate gRNAs were identified upstream of the gene of interest using CRISPR-P (crispr[dot]hzau[dot]edu[dot]cn) and selected for 1) high specificity to reduce the risk of off-target and 2) uniform distribution across the target site of interest. For target sites significantly greater than 300bp, gRNAs were determined every ~200-300bp. For target sites <300bp, gRNAs were spaced every ~50-150bp to ensure that at least two gRNAs target the locus of interest (e.g., 5'UTR). Tables 4 and 5 provide gRNA targets for OsVDE and OsZEP, respectively.

[0121] Eight gRNAs were assembled into a DNA cassette with interspersed scaffolds and tRNA linkers for polycistronic gRNA expression, as previously described (Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci. 2015; 112(11):3570-3575. doi:10.1073 / pnas.1420294112) for synthesis (Genscript). Inserts were cloned into the pRGEB32 rice Agrobacterium-mediated transformation vector (Addgene Plasmid #63142) via Golden Gate assembly for CRISPR / Cas9 editing by Agrobacterium-mediated rice transformation. Table 4: gRNA targets for promoter and 5'UTR of OSVDE (LOC_OS04G31040) Table 5: gRNA targets for promoter and 5'UTR of OsZEP (LOC_Os04g37619) Induction and transformation of embryonic callus

[0122] As described in Example 1, embryogenic callus of O. sativa ssp. japonica Nipponbare 2-4 (PsbS SOX allele described in Example 1) was prepared and transformed.

[0123] After transformation, T 0 Then, these T 0 Plants self-pollinate to produce T 1 The progeny were screened for differences in NPQ phenotype and gene expression as described in Example 1 to identify stable homozygous lines with the heritable editable allele. Example 3: Mutagenesis of non-coding sequences of Arabidopsis PsbS and VDE to identify effective target sites for overexpression

[0124] The editing method in Nipponbare (O. sativa ssp. Japonica) split the 8 original gRNAs across two sites distal (3-4 kb) or proximal (150-400 bp) to the OsPsbS1 start codon. Interestingly, the results described in Example 1 found that all 5 distal gRNA sites were non-essential - deletions across all 5 sites resulted in progeny with WT NPQ (e.g., event 25-4). Therefore, it is speculated that the 3 gRNAs within the 5'UTR and immediately upstream drive the observed phenotypic variation, including the significant overexpression of OsPsbS1 transcripts and proteins in Nipponbare event 2-4 (PsbS OX allele) of japonica rice subspecies (O. sativa ssp. japonica).

[0125] To determine whether mutagenesis of non-coding sequences (NCS, i.e., 5'UTR, 3'UTR, introns) is more promising in generating higher frequencies and / or greater degrees of overexpression, the NCS of the Arabidopsis thaliana PsbS (AtPsbS, At1g44575) and VDE (AtVDE, At1g08550) genes (whose downstream phenotypes can be rapidly resolved by chlorophyll fluorescence imaging) were edited. The relative activities of the mutant alleles generated in the stably edited lines were quantified to provide a reference for future design strategies for crops.

[0126] Interestingly, AtVDE is located in a gene-dense region and has no unique promoter, which can be mutagenized without disrupting neighboring genes. Similarly, the 5'UTR of AtPsbS is short (73 bp), with few possible gRNA targets for mutagenesis. Therefore, this multi-target approach will identify multiple pathways for overexpression, which can also expand the diversity of target sites for other species and genes when the genome structure is limited. Materials and methods Guide RNA design and construct cloning

[0127] CRISPR-P (crispr[dot]hzau[dot]edu[dot]cn) was used to identify guide RNA (gRNA) target sites within the NCS of AtPsbS and AtVDE, and 1) high specificity was selected to reduce off-target and 2) uniform distribution across the target sites of interest. For target sites significantly greater than 300bp, gRNA was determined every ~200-300bp. For target sites <300bp, gRNA was spaced every ~50-150bp to ensure that at least two gRNAs target the locus of interest (e.g., 5'UTR). Table 6 provides an overview of the fragment lengths and gRNA targets of the NCS mutagenesis of AtPsbS and AtVDE. Tables 7 and 8 provide gRNA targets for AtPsbS and AtVDE, respectively.

[0128] Eight gRNAs were assembled into DNA cassettes interspersed with scaffolds and tRNA linkers for polycistronic gRNA expression, as previously described (Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci. 2015; 112(11):3570-3575. doi:10.1073 / pnas.1420294112) for synthesis (Genscript). Inserts were cloned into pKI1.1R Arabidopsis thaliana Agrobacterium-mediated transformation vector (Addgene Plasmid #85808) via Golden Gate assembly for CRISPR / Cas9 editing by Agrobacterium-mediated transformation. Table 6: Fragment lengths and relative numbers of gRNA targets used for AtPsbS / AtVDE NCS mutagenesis Table 7: with promoter targeting controls gRNA targets for NCS mutagenesis of AtPsbS (At1g44575) Table 8: gRNA targets for NCS mutagenesis of AtVDE (At1g08550) Arabidopsis transformation and phenotypic analysis

[0129] WT Arabidopsis Col-0 was transformed with each of the seven constructs summarized in Table 6. T cells carrying Cas9 and gRNA for each target site (i.e., promoter, 5'UTR, 3'UTR, or intron) were collected. 1 Seeds were harvested and transformants were identified.

[0130] After selfing of the transformant lines, phenotype analysis was performed for stable, heritable, homozygous edited alleles across the region of interest, as described in Example 1. Phenotypic analysis identified differences in the abundance and magnitude of the edited overexpressed alleles across the different NCS target sites. Example 4: Non-coding sequence mutagenesis of PsbS in rice (Oryza Sativa), corn (Zea mays) and cowpea (Vigna Unguiculata)

[0131] This example describes non-coding sequence (NCS) mutagenesis in rice (Oryza sativa), maize (Zea mays), and cowpea (Vigna unguiculata) PsbS (ie, OsPsbS, ZmPsbS, and VuPsbS). Materials and methods Guide RNA design and construct cloning

[0132] Guide RNA (gRNA) target sites were identified within the NCS of OsPsbS, ZmPsbS, and VuPsbS as described in Example 3. The numbering of the gRNAs reflects the CRISPR-P output to track the top few candidate off-target loci. Tables 9, 10, and 11 provide gRNA targets for OsPsbS, ZmPsbS, and VuPsbS, respectively. For OsPsbS, the 5'UTR gRNA was identical to the two used in the initial construct to generate the PsbS OX allele described in Example 1.

[0133] Construct cloning was accomplished as described in Example 3. Table 9: gRNA targets for NCS mutagenesis of OsPsbS1 (Os01g64960) Table 10: gRNA targets for NCS mutagenesis of ZmPsbS (Zm00001d042697) Table 11: gRNA targets for NCS mutagenesis of VuPsbS (Vigun09g165900) discuss

[0134] In the simplest case, overexpression by CRISPR / Cas9 will be achieved by mutation or deletion of inhibitory cis-regulatory elements. However, the presence and maintenance of such repressors is likely determined by natural selection. It is speculated that CRISPR / Cas9 can also be used to drive the evolution of genes of interest (e.g., in non-coding sequences of PsbS) in order to generate novel genomic variants that produce overexpression phenotypes. It is further speculated that by editing the NCS of the gene of interest, the stability and half-life of the mRNA transcript can be increased, rather than increasing transcription of the gene, to increase the steady-state level of the mRNA, and therefore increase the level of protein translated from the mRNA. For example, the Nipponbare 2-4 overexpression allele generated in Example 1 may have 1) higher OsPsbS1 expression and / or 2) higher stability of the OsPsbS1 mRNA transcript to generate a novel overexpression phenotype.

[0135] Gao's team has previously shown that 5'-UTR editing to remove competing untranslated open reading frames (uORFs) in genes of interest can lead to overexpression phenotypes (Zhang et al., 2018, Nature Biotech, www[dot]nature[dot]com / articles / nbt.4202). However, the publication specifically emphasized that those edited genes did not have increased expression, but did have increased translation. It is predicted that this is due to the loss of ribosome competition during the translation of the gene of interest.

[0136] If CRISPR / Cas9 mutagenesis of NCS increases transcript stability rather than gene transcription, this approach and the specific sequence changes in Nipponbare 2-4OsPsbS1 OX are expected to be highly applicable to other species. It is also possible that specific sequence changes are transcript specific and thus require different sequence changes to overexpress other transcripts. A major bottleneck for future gene editing efforts will be the generation of similar NCS variants. As lead editing efficiencies increase and begin to become feasible in planta, known stabilizing sequences, such as those predicted to be within the 5'-UTR of OsPsbS1 overexpressed alleles, can be introduced into the 5-UTR of a gene of interest.

[0137] Enhancers and transcription stabilizers can be discovered by in planta / in vitro screening for promising candidates. For example, specific testing of a putative OsPsbS1 OX sequence in a plant expression vector (e.g., in Nicotiana benthamiana) can be used to verify the translatability of that particular sequence in grasses, monocots, and different plant species. Similarly, the experiments in A. thaliana described in Example 3 will also identify other promising candidates.

Claims

1. A method for screening a gain-of-function mutation in a target gene of a plant, the method comprising: (a) generating a set of mutations in a non-coding sequence (NCS) of a target gene in a population of plant cells of a plant using one or more RNA-guided nucleic acid modification enzymes targeted to a target gene, wherein the one or more RNA-guided nucleic acid modification enzymes targeted to a target gene comprise one or more different guide RNAs; (b) regenerating the population of plant cells into two or more plants that are hemizygous for the generated mutation; (c) (1) selfing two or more plants to produce progeny plants, and (2) optionally, screening progeny plants homozygous for the mutation for screening in part (d); and (d) screening the progeny plants from step (c) to identify gain-of-function mutations.

2. The method according to claim 1, further comprising: (e) selecting plants having a gain-of-function mutation, and (f) Sequencing of target genes to identify gain-of-function mutations.

3. The method of claim 1 or 2, wherein the one or more RNA-guided nucleic acid modification enzymes are expressed by an expression vector comprising a selectable marker, and the screening in step (d) comprises screening for plants lacking the selectable marker.

4. The method of claim 1 or claim 2, wherein the gain-of-function mutation induces overexpression of the target gene. The method according to claim 4 , wherein the overexpression of the target gene is in the morning. The method of claim 4 , wherein the overexpression of the target gene is not constitutive.

7. The method of claim 4, wherein the plant has a constitutive phenotype.

8. The method of any one of claims 1 to 7, wherein the target gene induces a phenotype associated with one or more of photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, and CO2 fixation, and the screening of step (c)(2) comprises screening progeny plants by chlorophyll fluorescence to identify non-transgenic plants that are putatively homozygous for the mutation.

9. The method of any one of claims 1-8, wherein the target gene induces a phenotype related to water use efficiency, and the screening of step (c)(2) comprises screening progeny plants by chlorophyll fluorescence to identify non-transgenic plants putatively homozygous for the mutation.

10. The method according to any one of claims 1 to 9, wherein the method does not comprise the use of plants having hypomorphic alleles or null alleles of the target gene.

11. The method of any one of claims 1-10, wherein the gain-of-function mutation improves yield, quality, or both in a plant having the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions.

12. The method of any one of claims 1-11, wherein the one or more RNA-guided nucleic acid modifying enzymes targeting a target gene comprise two or more different guide RNAs, three or more different guide RNAs, four or more different guide RNAs, five or more different guide RNAs, ten or more different guide RNAs, or twenty or more different guide RNAs.

13. The method of any one of claims 1-12, wherein each guide RNA targets a region of the target gene selected from the group consisting of a promoter region, an upstream regulatory region, a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), an intron, a micro-RNA binding site, an alternative splicing element, and a downstream regulatory element.

14. The method of claim 13, wherein the guide RNA targets the 5'UTR of the target gene.

15. The method of any one of claims 1-14, wherein the guide RNA targets at least a region in the target gene that is at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical across plant species.

16. The method according to any one of claims 1-15, wherein at least 50% of the mutation set is located in a region of the target gene selected from the group consisting of a promoter region, an upstream regulatory region, a 5'UTR, a 3'UTR, an intron, a micro-RNA binding site, an alternative splicing element, and a downstream regulatory element.

17. The method of claim 16, wherein at least 50% of the mutation set is located in the 5'UTR of the target gene.

18. The method of any one of claims 1-17, wherein the gain-of-function mutation is a deletion, inversion, translocation, insertion, transition, transversion, or a combination thereof.

19. The method according to any one of claims 1 to 18, wherein the gain-of-function mutation is an increase in transcription of the target gene, an increase in the stability of the mRNA produced by the target gene, an increase in the translation of the protein coding region of the mRNA, or a decrease in mRNA degradation, in each case compared to a plant lacking the gain-of-function mutation grown under the same conditions.

20. The method of any one of claims 1-19, wherein the one or more RNA-guided nucleic acid modification enzymes are Cas enzymes, base editors, or lead editors.

21. The method of claim 20, wherein the Cas enzyme is selected from the group consisting of Cas9, Cas12, Cas12a, Cas13, Cas14, CasX and CasY.

22. The method of any one of claims 1 to 21, wherein the plant is a crop plant, a model plant, a monocot, a dicot, a plant with Crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a maize plant, a biomass plant, an Arabidopsis thaliana plant, a tobacco (Nicotiana tabacum) plant, a rice (Oryza sativa) plant, a corn (Zea mays) plant, a sorghum (Sorghum bicolor) (sweet sorghum or grain sorghum) plant, a soybean (Glycine max) plant, a cowpea (Vigna unguiculata) plant, a poplar (Populus spp.) plant, a eucalyptus (Eucalyptus spp.) plant, a cassava (Manihot esculenta) plant, a barley (Hordeum vulgare) plant, a potato (Solanum tuberosum plants, sugarcane (Saccharum spp.) plants, alfalfa (Medicago sativa) plants, Miscanthus plants, energy sugarcane plants, elephant grass plants, wheat plants, oat plants, oil palm plants, safflower plants, sesame plants, flax plants, cotton plants, sunflower plants, camelina plants, rapeseed (Brassicanapus) plants, Ethiopian mustard (Brassica carinata) plants, mustard (Brassica juncea) plants, pearl millet plants, foxtail millet plants, other cereal plants, oilseed plants, vegetable crop plants, fodder crop plants, industrial crop plants or tree crop plants.

23. The method of claim 22, wherein the plant is a rice (Oryza sativa) plant, a corn (Zeamays) plant or a cowpea (Vigna unguiculata) plant.

24. A method according to claim 22 or claim 23, wherein the plant is an elite line or strain.

25. The method of any one of claims 1-24, wherein the target gene is selected from the group consisting of a photosystem II subunit S (PsbS) gene, a zeaxanthin epoxidase (ZEP) gene, and a violaxanthin de-epoxidase (VDE) gene.

26. The method of claim 25, wherein the screening comprises evaluating one or more of: photosynthetic efficiency under fluctuating light conditions; photoprotection efficiency under fluctuating light conditions; an increase in the rate of non-photochemical quenching (NPQ) induction under fluctuating light conditions; an increase in the rate of non-photochemical quenching (NPQ) relaxation under fluctuating light conditions; an improvement in quantum yield under fluctuating light conditions, and an improvement in CO2 fixation under fluctuating light conditions.

27. The method of claim 25 or claim 26, wherein the target gene is a PsbS gene, and wherein the PsbS gene comprises a sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:97 and SEQ ID NO:

98.

28. The method of claim 25 or claim 26, wherein the target gene is a ZEP gene, and wherein the ZEP gene comprises SEQ ID NO:

92.

29. The method of claim 25 or claim 26, wherein the target gene is a VDE gene, and wherein the VDE gene comprises a sequence selected from the group consisting of SEQ ID NO:92 and SEQ ID NO:

95.

30. The method of claim 27, wherein the one or more different guide RNAs comprise a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 The invention relates to an intervening sequence of SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:

90.

31. The method of claim 28, wherein one or more different guide RNAs comprise a spacer sequence selected from SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:

37.

32. The method of claim 29, wherein the one or more different guide RNAs comprise a spacer sequence selected from SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:

65.

33. The method of any one of claims 30-32, wherein one or more different guide RNAs are introduced using a vector, and wherein the vector comprises two or more gRNA scaffolds comprising SEQ ID NO: 9, and two or more tRNA linkers comprising SEQ ID NO:

10.

34. A method of producing improved cash crop plants or crop seeds, the method comprising: (a) selecting cash crop plants for improvement; (b) introducing a gain-of-function mutation identified in the method of any one of claims 1 to 33 into at least one cell of a cash crop plant to produce an improved cash crop plant cell; and (c) producing improved cash crop plants or crop seeds from improved cash crop plant cells.

35. The method of claim 34, wherein the cash crop plant comprises a rice (Oryza sativa) plant, a corn (Zea mays) plant, or a cowpea (Vigna unguiculata) plant.

36. An improved cash crop plant or crop seed comprising a gain-of-function mutation in a non-coding sequence of a target gene, wherein the target gene induces a phenotype associated with one or more of photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO2 fixation, and water use efficiency, and wherein the gain-of-function mutation improves the yield, quality, or both of the plant having the gain-of-function mutation compared to a plant lacking the gain-of-function mutation grown under the same conditions.

37. The improved cash crop plant or crop seed of claim 36, wherein the cash crop plant comprises a rice (Oryza sativa) plant, a corn (Zea mays) plant, or a cowpea (Vigna unguiculata) plant.

38. The improved cash crop plant or crop seed of claim 36 or claim 37, wherein the target gene is selected from the group consisting of a photosystem II subunit S (PsbS) gene, a zeaxanthin epoxidase (ZEP) gene, and a violaxanthin de-epoxidase (VDE) gene.

39. The improved cash crop plant or crop seed of claim 38, wherein the target gene is a PsbS gene, and wherein the PsbS gene comprises a sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:97, and SEQ ID NO:

98.

40. A genetically modified plant comprising an inversion in a cis-regulatory element of a PsbS gene, wherein the inversion increases expression of the PsbS gene.

41. The modified plant of claim 40, wherein the plant is a rice (Oryza sativa) plant.

42. The modified plant of claim 41, wherein the rice plant is an Oryza sativassp. japonica plant.

43. The improved plant of any one of claims 40-42, wherein the increased expression of the PsbS gene comprises overexpression, increased expression at one or more specific times, increased expression in one or more specific tissues, increased expression at one or more developmental stages, or a combination thereof, compared to a control plant without an inversion in the cis-regulatory element of the PsbS gene.

44. The modified plant of any one of claims 40-43, wherein the inversion encompasses a portion of the 5'UTR of the PsbS gene.

45. The improved plant of any one of claims 40-44, wherein the inversion comprises between 1,000 and 500,000 nucleotides, between 2,000 and 400,000 nucleotides, between 3,000 and 300,000 nucleotides, between 1,000 and 10,000 nucleotides, between 2,000 and 8,000 nucleotides, between 3,000 and 5,000 nucleotides, or about 4,000 nucleotides.

46. ​​The improved plant of any one of claims 40-45, wherein the plant or an ancestor thereof is screened for an inversion in the cis-regulatory element of the PsbS gene and increased expression of the PsbS gene.

47. The improved plant according to any one of claims 40 to 46, wherein the inversion in the cis-regulatory element of the PsbS gene arose randomly in the plant or an ancestor thereof.

48. The improved plant of claim 47, wherein the inversion is randomly generated using a guide RNA.

49. The improved plant of claim 48, wherein the guide RNA comprises a spacer sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:

90.

50. The improved plant of any one of claims 40-49, wherein increased PsbS expression results in a phenotype associated with one or more of photosynthetic efficiency, photoprotection efficiency, non-photochemical quenching, photosynthetic quantum yield, CO2 fixation, and water use efficiency.

51. The modified plant of claim 50, wherein the phenotype is constitutive.

52. The improved plant of any one of claims 40-51, wherein increased PsbS gene expression comprises overexpression of PsbS in the morning or is not constitutive overexpression.

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