PLANT BREEDING PARTNERSHIPS: Applications of CRISPR/Cas-mediated genome editing for precision breeding in wheat

PLANT BREEDING PARTNERSHIPS: APPLICATIONS OF CRISPR/CAS-MEDIATED GENOME EDITING FOR PRECISION BREEDING IN WHEAT

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1022329

Grant No.

2020-67013-30906

Cumulative Award Amt.

$650,000.00

Proposal No.

2019-05430

Multistate No.

(N/A)

Project Start Date

Jun 1, 2020

Project End Date

May 31, 2024

Grant Year

2020

Program Code

[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Plant Pathology

Non Technical Summary
Improving wheat yield potential to meet future demands for food while reducing land use and promoting sustainable farming requires concerted efforts of research and breeding community aimed at 1) discovering genetic variants controlling physiological and developmental processes that contribute to traits of agronomic importance, especially productivity and nutritional value traits, 2) evaluating the phenotypic effects of individual and stacked beneficial variants in adapted germplasm under realistic agricultural settings, and then 3) using this knowledge to redesign wheat plant by strategically combining those alleles whose interaction greatly enhances trait expression. While progress was made towards identifying the genetic basis of many agronomic traits, we still need to expand genetic diversity accessible for breeding by either introducing new diversity from the ancestral populations of wild relatives or landraces, or by creating novel variants by mutagenesis or gene editing. We still have limited understanding of how discovered genes and their alleles will perform in diverse genetic backgrounds or environments, or how traits affecting different aspects of wheat biology will interact with each other once combined. To address these challenges, urgent integration of innovative technologies and breeding strategies into the translational activities is required.Over the last decade, by the efforts of national and international wheat research programs, the genetic basis of many agronomic traits has been established. The release of the annotated wheat genome sequence and development of comparative genomics tools and resources provided a powerful framework for extrapolating gene mapping information from other crops into wheat. These discoveries now provide unique opportunity to redesign biological pathways underlying major agronomic traits in wheat by introducing the favourable alleles of causal genes into the breeding pipelines.Many studies demonstrated that the CRISPR-Cas system can take full advantage of these new genomic resources and facilitate the characterization and deployment of the novel gene variants. Our project will establish Plant Breeding Partnership including Kansas State University (KSU) and the University of Saskatchewan (Canada) with the aim to integrate the CRISPR-Cas-based technology into the wheat pre-breeding pipelines and improve wheat productivity and nutritional quality traits in adapted germplasm. This project will build on the resources generated in the on-going gene editing projects conducted by the project directors and collaborators. The project activities will be integrated with the Wheat CAP and IWYP projects, and the future NIFA IWYP Winter Wheat Breeding Innovation Hub in Manhattan (KS).Here, we will create variation in the protein coding or regulatory regions of genes that can influence plant growth, spike and grain development, and nutrient accumulation in grain. We will make use of CRISPR-edited wheat lines generated by our team in a previous project with mutations in the genes affecting grain size and weight, and number of grains per head.We will test a new strategy to create novel phenotypic variation by mutating the regulatory regions of genes controlling nitrogen uptake, carbon fixation, growth and nutrient remobilization. Currently, we have limited understanding of the range of possible quantitative variation for these traits that can be generated by editing the regulatory regions of genes. In this project, we will investigate phenotypes produced by the CRISPR-Cas9-edited promoter variants and assess their utility for wheat improvement. By combining and testing different regulatory variants of genes showing different levels of expression, we will identify optimal combinations of variants supporting effective nitrogen uptake and plant growth.The identification and assembly of positively interacting combinations of genes affecting wheat productivity traits has a potential to substantially increase yield. One of the pre-requisites for the implementation of this strategy is the identification of alleles that do not show negative interaction. By transferring the CRISPR-edited gene variants into the adapted germplasm already selected for other beneficial and complementary traits (e.g. high biomass, improved plant architecture, etc.), this project will investigate the utility of quantitative trait variation induced by gene editing for wheat improvement, and identify the combinations of germplasm and CRISPR-Cas induced alleles capable of suppressing negative interaction among the wheat production traits.With gene editing becoming one of the valuable assets in the breeder's crop improvement toolbox, training opportunities for graduate students and postdoctoral researchers that integrate gene editing system with breeding methodologies, bioinformatics, comparative genomics, and molecular genetics are required.In our project, we will develop educational modules for postdoctoral researchers and PhD students to provide training in applications of gene editing technology for crop improvement, regulatory aspects of crop biotechnology and science communication. These training is intended to prepare the new generation of crop scientists to face the challenges of communicating their discoveries to the public, consumer groups and policymakers to ensure that the science-based policies prevail.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1549	1080	80%
201	1549	1081	20%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

gene editing

translational research

wheat breeding

yield

Goals / Objectives
Overall goal: Our goal is to engineer novel variants of genes and their combinations in the adapted germplasm and test their effects on the wheat productivity and quality traits with the long term goal of establishing the CRISPR-Cas-based technology as a precision breeding tool for wheat improvement.The specific objectives of the project:1) Identify traits and genes for CRISPR-Cas-assisted introgression into adapted wheat cultivars; 2) Broaden phenotypic diversity of bread wheat by engineering the allelic series of the promoters of genes controlling wheat productivity and nutritional quality traits;3) CRISPR-Cas-assisted engineering and transfer of gene variants into adapted germplasm;4) Evaluate phenotypic effects of gene editing in the adapted germplasm for development, yield component and nutritional quality traits;5) Expand training opportunities for the next generation of scientists and breeders in genome editing strategies.

Project Methods
Selection of genes for CRISPR editingIn the on-going NIFA IWYP-funded project, we have identified and edited 16 genes that can positively influence yield component traits in wheat, such as grain size, grain weight, and grain number per spike. For five of these edited variants of genes (TaGW2, TaGW7, TaCKX2-1, TaGW8, TaGASR7, TaCKX2-3), our group or other published studies have confirmed positive effect on the phenotypes of interest. The natural allelic variation in many other genes was shown to have effect on yield component traits in the diversity panels, suggesting that edited variants of these genes will likely have positive effects on yield-related traits. We will test whether the introduction of our edited yield component genes (mostly affecting sink-related traits) into the germplasm expressing exceptional source-related traits (high biomass, beneficial root architecture, high spikelet number per spike) will improve wheat yield potential.We also selected three wheat genes (Rht1, TaGRF4 and TaNAM-B1) controlling plant growth, metabolism of carbon and nitrogen, and nutritional quality traits for promoter editing using CRISPR-Cas technology. Using multiplex CRISPR-based genome editing, we will create allelic series of these genes.Design and testing of gRNA constructsTo design single guide RNA (sgRNA) constructs for Cas9 and Cpf1 endonucleases, we will combine bioinformatical design using the sgRNA Scorer 2.0 and CRISPR-DT with the high-throughput screening of sgRNAs using the wheat protoplast assay. To select gRNAs with no off-target activity, the high-scoring sgRNAs will be compared against the wheat reference genome. For targeting the gene promoters, we will design up to 20 sgRNAs targeting regions spanning 2-3 kb upstream of the gene. The editing strategies requiring the simultaneous expression of multiple sgRNAs will be implemented using the multiplex gene editing constructs where sgRNAs are separated by the tRNA spacers. All constructs will be tested in the wheat protoplasts followed by the NGS of target sites.Generation of transgenic plantsTo generate populations of wheat lines carrying different promoter alleles, T0 generation plants expressing gRNA constructs will be crossed with the wild-type plant. Due to trans-generational activity, the CRISPR-Cas9-gRNA transgenes will target in trans the wild type promoters, producing various allelic variants in the progeny of F1 plants. The F2 generation plants will be screened for editing events by the NGS of multiplexed amplicons spanning promoter regions. Two spring wheat cultivars 'Fielder' and 'Bobwhite' will be used for transformation. Mutations in the targeted regions will be identified by the NGS of barcoded PCR amplicons.Transfer of gene editing events to the recipient germplasmAdapted germplasm for transferring CRISPR-edited gene variants will be selected based on the nature of affected traits. Most of the edited genes in our on-going project influence sink-related traits (grain size/weight, number of grains per spike). We will transfer multiple edited variants of these genes into the same cultivar that also shows superior source-related traits (high root biomass or above ground biomass). To transfer gene editing events to recipient cultivar, we will use tested a strategy based on crossing cv. Bobwhite expressing Cas9 transgene at high levels with a recipient germplasm. This approach allowed for efficient gene editing of the recipient's gene copies in the F1 hybrids. The recurrent parent genome (RPG) will be recovered by two rounds of backcrossing to recipient parent (recover 87.5% of RPG), each time selecting for the presence of CRISPR-Cas-gRNA constructs, and gene editing mutations in the targeted gene.Promoter editing: T0 generation Bobwhite or Fielder expressing the multiplexed CRISPR-Cas-gRNA constructs at high level will be identified and crossed with the recipient germplasm. The F2 progeny will be screened to identify promoter deletions in the recurrent parent's gene copy. The lines with deletions will be selected, and backcrossed twice with the recurrent parent, at each step selecting for the presence of promoter deletions and absence of Cas construct.Phenotypic evaluationPlant productivity traits: Transgenic lines carrying the edited version of genes will be grown in both greenhouse and field conditions. For phenotyping in the greenhouse, plants will be grown for 16 h light / 8 h dark. While plants with validated mutations will be also phenotyped at T1 and T2 generation, the effects of gene editing on phenotype will be assessed on BC2F2:3 plants in greenhouse. Seedlings of BC2F2 will be grown in 96-well trays, genotyped, and at least 20 plants with and without homozygous Cas-induced mutations will be transplanted to 1L pots and phenotyped. Phenotypes of BC2F2:3 plants will be confirmed in the next generation by growing 10 plants from at least 10 families within each group (presence or absence of mutations). Experiment will be organized according to complete randomized design, as described.Field phenotyping of spring wheat lines will be performed on BC2F4:5 lines grown in Davis (CA) and Saskatoon (Canada). Entries will be planted in the randomized complete block design in three replicates in 4-row 2 m-long plots (260 seeds per m2). For some of the yield component traits we have already initiated crosses to transfer gene edits to the adapted germplasm.Standard phenological data such as timing of stem elongation, date of heading, date to anthesis, date of physiological maturity, and plant height will be collected. Yield-related phenotypes collected will include yield and test weight, as well as yield components including thousand grain weight, the number of fertile tillers/spikes per plant or per unit area, average number of kernels per spike, and average kernel weight.Nutritional quality traits: Senescence will be estimated by measuring non-destructively the relative chlorophyll content in the flag leaf of each main spike using a hand-held chlorophyll meter. Nitrogen concentration in ground tissues will be measured by NIR reflectance using a Perten DA7250 calibrated to combustion analysis. Ionomic analyses to assess the Fe and Zn concentrations in grain and flag leaves will be conducted by the facility in the Donald Danforth Plant Science Center.For each trait, we will estimate the Best Linear Unbiased Estimates (BLUEs) from a spatial model using the ASReml package in R following the previously described procedures. A model is flexible and can include the effects of environment, year, experiment design, followed by fitting the best spatial model to genotype. The BLUEs of each phenotypic trait obtained for lines with and without gene editing mutations will be compared to assess the effect of gene editing on wheat productivity and nutritional quality traits. Workshops Our project will develop educational modules for postdoctoral researchers and PhD students covering the basics of the gene editing technology. For this purpose, we will work with the KSU Integrated Genomics Facility, on incorporating gene editing training modules into the summer workshops for PhD students (2 credit course) organized on the yearly basis by the KSU IGF. Three summer workshops will be co-taught by the project team including PhD students, postdoctoral researchers, PDs and co-PDs in collaboration with the IGF personnel. The IGF routinely will conduct on-line course evaluation and provide feedback to the project team. A graduate student from the co-PD Pozniak's group will visit KSU campus each summer to participate in the workshops and discuss project activities.

Progress 06/01/20 to 05/31/24

Outputs
Target Audience:Our target audiences are 1) stakeholders, wheat breeders, farmers, government, and industry representatives whose feedback play important role in selecting targets for genome editing project; 2) students and postdoctoral researchers who are interested in application of the genome editing technology in breeding and research; 3) general public whose opinion influences the acceptance of gene editing technology in breeding and development of new crop varieties. Changes/Problems:The project was substantially affected by COVID19, which coincided with the departure of two main researchers on the project. These events delayed progress with the development and evaluation of gene edited lines. In addition, the editing efficiency of multiplex editing for creating mutations in the promoter regions turned out to be relatively low. Only with establishing the virus-based system of gRNA delivery, we managed to overcome this limitation. The BSMV system was critical for generating GRF4 promoter mutants, which showed variation in major agronomic traits affecting productivity and grain quality. Since these developments occurred only during the third year of the project, generation of promoter mutants for NAM-B1 (now at M1 generation) was delayed. The evaluation of NAM-B1 mutants will be outside of the project timeframe. What opportunities for training and professional development has the project provided?Postdoctoral researcher Wei Wang along with PD E. Akhunov participated as mentors in 8-week KSU Summer 2022 internships in plant health at Kansas State University. Undergraduate student Nicholas Stelling (Iowa State University) joined PD Akhunov Lab to work on a gene editing research project. He learned 1) how to edit wild tetraploid wheat lines expressing Cas9 construct by inoculating them with BSMV-gRNA transcripts, 2) how to detect gene editing events by next-generation sequencing, and 3) how to use KASP genotyping markers to trace introgression of wild relative alleles in adapted wheat cultivars. At the end of REU internship, Nick presented a poster entitled "Development of strategies to improve modern wheat cultivars by adopting genetic diversity from wild relatives" at the Summer Undergraduate Research Showcase (July 28, 2022, Kansas State University, Manhattan, KS). Nicholas Stelling will be joining our group in spring of 2023 as a PhD graduate student. Research and mentoring experience gained by postdoctoral researcher Wei Wang (PD E. Akhunov group) in the USDA NIFA-funded project were critical for him to receive a faculty position in the Nanjing Agricultural University (China), one of the top agricultural research centers focused on crop research and breeding. His future research will be focused on application of gene editing to improve wheat. Two postdoctoral researchers (Wei Wang, Zitong Yu) involved into the project have attended the workshop "Barley and Wheat Transformation" organized by the Bill and Melinda Gates Foundation (June 15th, 2021). A postdoctoral researcher Z. You presented her work on editing the promoter regions of transcription factors controlling plant development at the Plant and Animal Genome 31 conference (January 12 - January 17, 2024. San Diego, CA, USA). Nine undergraduate students from Kansas State University have been involved into the gene editing research project (Taiwo Bamidele, Kynlie Ibarra, Yunxia (Rebecca) Zhang, Samantha Webber, Sabreena Leach, Zach Rosenkrans, Matthew Walter Long, Honoree Massonne Koumba, Joshua Anthony Russell), and receive training in phenotyping and genetic characterization of gene editing plants. How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered presentations at the meetings organized by the Kansas Association of Wheat Growers, Kansas Wheat Commission (KWC), and Heartland Plant Innovations Center (HPI) and K-State Research and Extension: 1) The U.S. Wheat Associates International Staff Training, March 28, 2022, Manhattan, KS. 2) Kansas Wheat Research Committee. April 20, 2022, Manhattan, KS. 3) U.S. Wheat Associates World Staff Conference, Maui, Hawaii, Aug. 23, 2022. 4) Bay State Milling Meeting, KWIC, Manhattan, KS, May 31, 2023. 5) Meeting with the Bimbo Bakeries technical group. Kansas Wheat Innovation Center, Manhattan, KS. April 19, 2023. We worked with the U.S. Wheat Associates, the National Association of Wheat Growers (NAWG), and the National Wheat Improvement Committee (NWIC) to comment on recently published proposed exemptions concerning the Movement of Organisms Modified or Produced through Genetic Engineering (APHIS-2023-0022-0001). These comments were intended to express concerns about the proposed exemptions that largely preclude allopolyploids in general and wheat in particular from participating in the use of gene editng technology. Wheat researchers, breeders, industry representatives: The outputs of the project were disseminated to the international wheat community by publishing in the international peer-reviewed journals. Project results are also presented to wheat researchers, breeders and industry by preparing a press release in coordination with International Wheat Yield Partnership. The project team presented their discoveries to wheat researchers and breeders at the national and international conferences: 1) BreedWheat Symposium: Advances and Beyond, 2-4 September, 2020, Clermont-Ferrand, France, 2) 2020 ASA-CSSA-SSSA International Annual Meeting, November 9-13, 2020. 3) The results of this gene editing project were also included into the talk presented at the International Wheat Genome Sequencing Consortium Fall 2020 Webinar Series, Oct. 7. 4) USDA NIFA Project Director Meeting at the National Association of Plant Breeders. 2023 Annual Meeting. Clemson University. Greenville, SC; 5) 7th International Wheat Yield Partnership Program Conference, 27 & 28 September 2023; 6) Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA. 7) Plant and Animal Genome 31 Conference. January 12-17, 2024. San Diego, CA, USA. 8) The press release about the gene editing project was prepared in collaboration with the IWYP team (IWYP Science Brief No. 17, July 2021; https://iwyp.org/iwyp-science-briefs/). The outputs of the project are disseminated to the international wheat community by publishing in an international peer-reviewed journal. Distribution of wheat lines and plasmid constructs: The wheat lines with high Cas9 expression and BSMV constructs have been distrusted to multiple research labs. Students and postdoctoral researchers: The KSU team participates in the two-month REEU program run the KSU Department of Plant Pathology by hosting an undergraduate student (Nick Stelling). PD Akhunov delivered a lecture at the Genomic Technologies Workshop (Kansas State University, Manhattan, KS, June 6-9, 2022). Also, Dr. Wang delivered two presentations covering our wheat gene editing project to graduate students and young researchers: 1) Wang W. CRISPR-Cas genome editing technology as a tool for precision breeding in wheat, Graduate seminar class series, Department of Plant Sciences, University of Idaho, Moscow, ID, Oct. 22, 2021; 2)Wang W.Development and Application of High-Throughput Gene Editing Technology in Wheat, 5th Zhongshan International Young Scientist Symposium, Nanjing Agriculture University, Nanjing, China, May 28, 2022. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Broadening the wheat genome editing toolbox Multiplex editing system based on Cpf1 (Cas12a): We have expanded the range of editable targets in the wheat genome by developing a Cas12a-based editing platform. This platform also offers a higher level of multiplexing compared to the traditional Cas9 system. We tested Cas12a for multiplex gene editing (MGE) in wheat and successfully obtained mutations in the GASR7, GW7, An1, SPL16, GW2, GS3, and GSE5 genes. This study was published in the Plant Biotechnology Journal (Wang et al., 2021). BSMV-based editing: We have developed a virus-based gene editing system for wheat to create mutations in the regulatory regions of target genes. In a study published in the Plant Biotechnology Journal (Wang et al., 2022), we used pooled BSMV-sgRNAs to create deletions in the Q gene promoter. We have since adopted BSMV-based editing to generate regulatory mutations in TaGRF4, targeted in our project (see below), and for editing domestication genes and targeting the regulatory regions of NAM-B1 and SPL14 TFs in another USDA NIFA-funded project. Editing the regulatory regions GRF4 transcription factor: The GRF4 and Rht-1 genes have opposing effects on plant growth, nitrogen uptake, and carbon fixation, with GRF4 acting as a positive regulator. We targeted the GRF4 promoter region and miR396 site in three homoeologs on chromosomes 6A, 6B, and 6D. The gRNAs were cloned into the BSMV construct for delivering into Bobwhite cultivar expressing Cas9. We identified six M1 lines carrying indels in the promoters of the GRF4 homoeologs: 10-13 (106 bp deletion on 6A), 10-19 (89 bp deletion on 6B), 10-67 (93 bp deletion on 6D), 5-5 (103 bp deletion on 6A), 10-58 (83 bp deletion on 6D), and CRS1-65 (108 bp insertion on 6D). The effects of these cis-regulatory mutations on gene expression and phenotype were evaluated in homozygous M3 lines. RNA extracted from leaves at the Z10 stage was used to compare expression between wild-type (non-edited) and mutant lines. Compared to wild-type plants, all mutants showed significant changes in the expression of at least one GRF4 homoeolog. Three mutants with edits on the 6D chromosome (10-67, CRS1-65, 10-58) showed up-regulation of total GRF4 expression, whereas one mutant (10-13) with a 6A chromosome edit showed down-regulation of total GRF4 expression. In one of the 6D homoeolog mutants (10-67), statistically significant up-regulation of the 6D homoeolog coincided with down-regulation of the 6B homoeolog, suggesting compensatory mechanisms maintaining expression balance between the wheat genomes. The wild-type and M3 mutant plants were phenotyped for tiller number, plant height, peduncle length, spike length, spikelet number, grain number, grain weight, grain area, grain width, grain length, and grain protein content (GPC). Compared to the wild-type, mutant 10-67 showed substantially reduced tiller number, while mutants 5-5 and 10-58 showed significantly increased tiller numbers. Three mutants (10-13, 10-19, 10-58) showed significant increases in plant height (up to 13%) and peduncle length (up to 16%). Mutant 5-5 did not show a substantial change in plant height but had reduced peduncle length. Mutants 10-67 and 5-5 showed reduced and increased spike length/spikelet number, respectively. Mutants 10-13 and 10-19, with edits on chromosomes 6A and 6B, both showed down-regulation in GRF4 expression, leading to increased grain number (9-11%) and grain weight (9-14%). This increase was accompanied by a slight 7% decrease in GPC relative to wild-type plants in mutant 10-13, with no changes in GPC in mutant 10-19. Mutant 5-5 also showed a significant increase (7%) in grain number and grain weight without negatively impacting GPC. Mutant 10-67, with up-regulated GRF4, showed a significant reduction in grain number (18%) and grain weight (25%) but an increase in GPC (13% relative to wild-type). Mutant 10-58 showed a reduced GPC without substantial changes in grain number or grain weight. The GRF4 cis-regulatory mutants showed variation in grain morphometric traits. Mutant 10-13 had an increased grain width, while mutant 10-67 showed a decrease in grain width, length, and area. Mutants 10-13, 10-19, and 10-58 had reduced grain lengths. Other mutants did not show significant changes in grain morphometric traits. Notably, mutant CRS1-65, despite showing changes in GRF4 expression due to an insertion in the promoter region, did not show statistically significant changes in any of the investigated traits. This suggests that the observed changes in specific phenotypic traits are associated with mutations in cis-acting elements within the promoter region rather than changes in GRF4 expression levels. In summary, we have created a series of cis-regulatory mutants of the GRF4 transcription factor that show variation in major developmental, quality, and yield component traits. These mutants broaden the set of beneficial alleles for improving wheat in breeding programs. Editing miR396 site: To edit the GRF4 miR396 site, we designed two sgRNAs and used BSMV-based delivery systems to modify Bobwhite. The M0 plants GRF4T4-1/11 with high editing efficiency (39.4%) and GRF4T4-6/16 with a 6-bp in-frame deletion are used to make M1 population to identify homozygous miR396 mutants. The M1 and M2 generation plants will be analyzed to study the effects of miR396 site editing on GRF4 expression and agronomic traits. NAM-B1 promoter editing: The two promoter mutants were generated for PI 664549 with the domesticated allele of NAM-B1. The multiplexed gRNAs were delivered using BSMV into PI671999 that expresses Cas9 and carries wild NAM-B1 allele. The M1 generation seeds of seven BSMV-inoculated plants have been planted and being screened for novel promoter mutations. CRISPR editing of negative regulators of grain number, size and weight TWG6 gene editing: We have edited a group of four TaTGW6 genes from chromosomes 3A (2 copies), 3B (1 copy), and 3D (1 copy). The 5191-2 line, which had all four TGW6 copies mutated, was used to develop 200 RILs showing variation in TGW6 dosage. Phenotyping for yield-related traits showed that an increase in the number of edited genes is associated with increased grain size and weight (up to 4%) without affecting grain number. ARF4 gene editing: ARF4 is a negative regulator of grain size. By characterizing T1 population derived from a line with a knockout mutation in the B genome, we observed 2.5% increase in grain weight. This result was confirmed in T2 population, which showed 3.2% increase in grain weight. Analysis of F3 generation lines derived from a cross between Bobwhite and T0 plant 7388-1, which carries triple knockout mutations in both copies of ARF4, confirmed the results prior analyses. Introgression of edited alleles into adapted cultivars: We have introgressed and evaluated the effects of editing in GW2, ARF4 and CKX2 gene in cultivars OK16D101089 and Bob Dole. The positive effects on grain size (3-5%), weight (3-8%) or number of grains per spike (3-5%) was confirmed for the edited allelic variants genes in the 2023-2024 greenhouse experiment. International partnership with the University of Saskatchewan We have established a partnership with the Canadian wheat genomics and breeding program led by C. Pozniak. In this project, the CRISPR-Cas9 system is used for editing genes controlling domestication and adaptive traits (BTR1, Q, Ppd1, VRN1, and Rh-B1) to condition non-adapted genetic lines, including wild relatives, for deployment in wheat breeding programs. The team conducted direct editing of domestication genes in wild emmer TG3487, utilizing the GRF4-GIF vector. Transformation and sequencing analysis of T0 plants demonstrated successful editing at all five target genes, although not within a single plant. In addition, a wild emmer accession expressing Cas9 was developed and successfully used for editing the Q gene using the BSMV-based gRNA delivery system.

Publications

Type: Journal Articles Status: Published Year Published: 2024 Citation: Wang W, Yu Z, Pan Q, Tian B, Davidson D, Bai G, Akhunova A, Trick H, Akhunov E. Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat. BioRxiv, 2024. doi: https://doi.org/10.1101/2024.04.28.591550
Type: Other Status: Published Year Published: 2024 Citation: Stelling N. Identification and editing of domestication genes in wheat. Student proposal seminar series, Plant Pathology, KSU Manhattan, KS. April 3, 2024.
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yu Z, Akhunova A, Trick H, Akhunov E. Application of Multiplex Genome Editing Strategies for Editing Regulatory Regions of the Wheat Genome. Plant and Animal Genome 31 Conference. January 12-17, 2024. San Diego, CA, USA.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Akhunov E. CROP BREEDING INNOVATION HUB: NIFA IWYP Winter Wheat Breeding Innovation Hub at Kansas State University 7th IWYP Program Conference (Zoom), 27 & 28 September 2023.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Akhunov E. CRISPR-based editing of wheat genome for gene discovery and broadening phenotypic diversity. USDA NIFA Project Director Meeting at the National Association of Plant Breeders. 2023 Annual Meeting. Clemson University. Greenville, SC, July 16-20. 2023.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Our target audiences are 1) stakeholders, wheat breeders, farmers, government, and industry representatives whose feedback play important role in selecting targets for genome editing project; 2) students and postdoctoral researchers who are interested in application of the genome editing technology in breeding and research; 3) general public whose opinion influences the acceptance of gene editing technology in breeding and development of new crop varieties. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher Z. You presented her work on editing the promoter regions of transcription factors controlling plant development at the Plant and Animal Genome 31 conference (Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA.). Seven undergraduate students from Kansas State University have been involved into the gene editing research project (Yunxia (Rebecca) Zhang, Samantha Webber, Sabreena Leach, Zach Rosenkrans, Matthew Walter Long, Honoree Massonne Koumba, Joshua Anthony Russell), and receive training in phenotyping and genetic characterization of gene editing plants. How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered three presentations at the meetings organized by the Kansas Association of Wheat Growers, Kansas Wheat Commission (KWC), and Heartland Plant Innovations Center (HPI) and K-State Research and Extension. 1) Bay State Milling Meeting, KWIC, Manhattan, KS, May 31, 2023. 2) Meeting with the Bimbo Bakeries technical group. Kansas Wheat Innovation Center, Manhattan, KS. April 19, 2023. Wheat researchers, breeders, industry representatives: The project team presented their discoveries to wheat researchers and breeders at the national and international conferences: 1) 7th International Wheat Yield Partnership Program Conference, 27 & 28 September 2023; 2) Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA. What do you plan to do during the next reporting period to accomplish the goals?The highly replicated phenotypic evaluation of the M3 generation of GRF4 promoter mutants will be performed in 2023-2024. The results of gene expression analyses and phenotyping for developmental, quality and yield component traits GRF4 promoter mutants will be reported. In 2023-2024, we will finalize genotypic and phenotypic evaluation of wheat cultivars carrying CRISPR-induced mutations in TaAFR4, TaCKX2-1, TaCKX2-2, and TaGW2 that affect sink-related traits (grain size, weight and number).

Impacts
What was accomplished under these goals? Editing the regulatory regions of genes controlling productivity traits NAM-B1 promoter editing: In 2022, we have identified developed the T0 Bobwhite lines each expressing three multiplexed gRNA constructs (3 gRNA/per construct). These lines were crossed with spring wheat PI671999 that carries the functional wild-type allele of NAM-B1. The F1 lines carrying the Cas9 constructs were backcrossed to PI671999 and used to developed BC1F2 population. Currently, we are screening the BC1F2 population for cis-regulatory mutations in the NAM-B1 promoter region. To increase our chances of recovering the cis-regulatory mutations, the BC1F2 lines expressing Cas9 constructs were identified to be used for BSMV-based inoculation with gRNAs targeting the promoter region of NAM-B1. GRF4 promoter editing: To create new variants of GRF4 with distinct levels of expression and growth phenotypes, we targeted the GRF4 promoter region (4 sgRNAs designed to regions conserved among homoeologs) and microRNA396 binding sites. Each gRNA targeting these regions was cloned into the BSMV construct, which was used to deliver the gRNAs into cultivar Bobwhite expressing Cas9. A total of 58 M1 lines (17%) derived from BSMV-inoculated plants targeting GRF4 promoter carried mutations in at least one homoeologous copy. We identified small scale deletions ranging from single base to tens of base pairs at the sites targeted by each of the 4 sgRNAs. We also identified six lines that carry deletions of the promoter regions between two sgRNA target sites. These lines were used to develop the M2 generation population, which was genotyped and used for preliminary phenotypic evaluation of productivity and developmental traits. Based on these results, we selected six homozygous cis-regulatory mutants for developing M3 generation population including at least 40 replicates of each genotype. Phenotypic evaluation of this population will be completed in the spring of 2024. Introgression of gene edited alleles into winter wheat We have developed BC1F2 and BC2F2 populations with introgression of the edited alleles of GW2, CKX2, ARF4, and GW7 into winter wheat cultivars Bob Dole, KS090387K-20 and IDO676. The genotyping and phenotyping of these lines for grain size, weight and number is currently underway. ARF4 gene editing ARF4 is a negative regulator of grain size. By characterizing T1 population derived from a line with a knockout mutation in the B genome, we observed 2.5% increase in grain weight. This result was confirmed in T2 population, which showed 3.2% increase in grain weight. Analysis of F3 generation lines derived from a cross between Bobwhite and T0 plant 7388-1, which carries triple knockout mutations in both copies of ARF4, confirmed the results prior analyses.

Publications

Type: Journal Articles Status: Published Year Published: 2023 Citation: Yu Z, Yunusbaev U, Fritz A, Tilley M, Akhunova A, Trick H, Akhunov E. CRISPR-based editing of the ?- and ?-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality. Plant Biotechnol J. Plant Biotechnol J. 2023 Nov 17. doi: 10.1111/pbi.14231.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yu Z, Wang W, Yunusbaev U, Fritz A, Tilley M, Akhunova A, Trick H, Akhunov E. Application of Multiplex Genome Editing Strategies for Engineering Regulatory Regions and Complex Gene Loci in Wheat. Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: He F, Wang W, Jordan K, Fritz A, Hayden MJ, Akhunova A, Dubcovsky J, Morrell PL, Szabo L, Rouse M, Akhunov E. Uncovering the Regulatory Landscape of the Wheat Genome and Its Connection with Agronomic Trait Variation. Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Our target audiences are 1) stakeholders, wheat breeders, farmers, government, and industry representatives whose feedback play important role in selecting targets for genome editing project; 2) students and postdoctoral researchers who are interested in application of the genome editing technology in breeding and research; 3) general public whose opinion influences the acceptance of gene editing technology in breeding and development of new crop varieties. Changes/Problems:Special circumstances Due to COVID-19 related situation, lab closure and stay-in-home policy, production of transgenic plants and their phenotypic evaluation was significantly delayed in 2021 and impacted experiments planned for 2022. Due to COVID-19 we also experience difficulties with recruiting new graduate students and technical personnel. What opportunities for training and professional development has the project provided?Postdoctoral researcher Wei Wang along with PD E. Akhunov participated as mentors in 8-week KSU Summer 2022 internships in plant health at Kansas State University. Undergraduate student Nicholas Stelling (Iowa State University) joined PD Akhunov Lab to work on a gene editing research project. He learned 1) how to edit wild tetraploid wheat lines expressing Cas9 construct by inoculating them with BSMV-gRNA transcripts, 2) how to detect gene editing events by next-generation sequencing, and 3) how to use KASP genotyping markers to trace introgression of wild relative alleles in adapted wheat cultivars. At the end of REU internship, Nick presented a poster entitled "Development of strategies to improve modern wheat cultivars by adopting genetic diversity from wild relatives" at the Summer Undergraduate Research Showcase (July 28, 2022, Kansas State University, Manhattan, KS). Nicholas Stelling will be joining our group in spring of 2023 as a PhD graduate student. Research and mentoring experience gained by postdoctoral researcher Wei Wang (PD E. Akhunov group) in the USDA NIFA-funded project were critical for him to receive a faculty position in the Nanjing Agricultural University (China), one of the top agricultural research centers focused on crop research and breeding. His future research will be focused on application of gene editing to improve wheat. Seven undergraduate students from Kansas State University have been involved into the gene editing research project (Yunxia (Rebecca) Zhang, Samantha Webber, Sabreena Leach, Zach Rosenkrans, Matthew Walter Long, Honoree Massonne Koumba, Joshua Anthony Russell), and receive training in phenotyping and genetic characterization of gene editing plants. How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered three presentations at the meetings organized by the Kansas Association of Wheat Growers, Kansas Wheat Commission (KWC), and Heartland Plant Innovations Center (HPI) and K-State Research and Extension. 1) The U.S. Wheat Associates International Staff Training, March 28, 2022, Manhattan, KS. 2) Kansas Wheat Research Committee. April 20, 2022, Manhattan, KS. 3) U.S. Wheat Associates World Staff Conference, Maui, Hawaii, Aug. 23, 2022. Wheat researchers, breeders, industry representatives: The outputs of the project are disseminated to the international wheat community by publishing in an international peer-reviewed journal. Students and postdoctoral researchers: The KSU team participates in the two-month REEU program run the KSU Department of Plant Pathology by hosting an undergraduate student (Nick Stelling). PD Akhunov delivered a lecture at the Genomic Technologies Workshop (Kansas State University, Manhattan, KS, June 6-9, 2022). Also, Dr. Wang delivered two presentations covering our wheat gene editing project to graduate students and young researchers: 1) Wang W. CRISPR-Cas genome editing technology as a tool for precision breeding in wheat, Graduate seminar class series, Department of Plant Sciences, University of Idaho, Moscow, ID, Oct. 22, 2021; 2)Wang W.Development and Application of High-Throughput Gene Editing Technology in Wheat, 5th Zhongshan International Young Scientist Symposium, Nanjing Agriculture University, Nanjing, China, May 28, 2022 What do you plan to do during the next reporting period to accomplish the goals?For NAM-B1 promoter editing, we will generate the T0 plants in the background of PI 664549 line carrying the wild allele of the NAM-B1 gene. These lines will be transformed with all three multiplexed editing constructs (gRNA-Cas9-GRFGIF-Bar) and evaluated for the presence of cis-regulatory mutations. An alternative NAM-B1 promoter editing strategy will be based producing PI 664549 line with the introgression of Cas9 locus. In 2023, we will initiate evaluation of the phenotypic consequences of NAM-B1 promoter editing on agronomic traits. The phenotypic evaluation of the GRF4 promoter mutants will be performed in 2022-2023. To generate mutations in the microRNA396 binding site, the progeny of M0 and T0 plants showing more than 10% editing efficiency will be evaluated. In the M1 and T1 plants, the expected editing events with the mutations in the microRNA396 binding site will identified by NGS. In addition, we will test the ability of Prime Editing technology to generate mutations at the miRNA binding site. In 2022-2023, we will finalize genotypic and phenotypic evaluation of wheat cultivars carrying CRISPR-induced mutations in single (TaAFR4, TaCKX2-1, TaCKX2-2, TaTGW6, TaGS3) and multiple genes that affect sink-related traits (grain size, weight and number). These wheat cultivars were selected based on the presence of natural alleles at gene loci controlling both source- and sink-related traits and distinct from gene loci subjected to CRISPR editing. In 2023, the KSU team will participate in KSU REU internship program. The KSU PDs and postdoctoral researchers will also contribute to developing the gene editing training modules for the annual genomics technologies workshop organized by the KSU Integrated Genomics Facility.

Impacts
What was accomplished under these goals? Broadening the wheat genome editing toolbox Creating new cis-regulatory diversity using a BSMV-based method of gRNA delivery: CRISPR/Cas9-induced cis-regulatory mutations in genes controlling productivity traits could broaden range of phenotypic variation and help to overcome the negative impact of epistasis on major agronomic traits. However, application of this strategy to mining beneficial cis-regulatory diversity remained limited due to the difficulties associated with production of cis-regulatory mutants. In our project, we demonstrate that BSMV-based viral system is an effective tool for creating new allelic variants in the regulatory regions of the wheat genome. This study was published in the Plant Biotechnology Journal (Wang et al., 2022). We used pooled BSMV-sgRNAs to create deletions in the promoter of the Q gene allele on chromosome 5A, which affects spike morphology, free-threshing habit, rachis fragility, spike length, number of spikelets per spike, and number of grains per spikelet and spike. We identified five M1 plants that had deletions in the homozygous or heterozygous state or present as mosaic somatic mutations. Two of these plants were homozygous for deletions of the targeted promoter regions. Our results showed that BSMV-based gRNA delivery could be an effective tool for creating new regulatory diversity for agronomic genes. We switched to the BSMV-based gRNA delivery system for generating the regulatory mutations in the TaGRF4 and NAM-B1 genes targeted in our project (see below). Agrobacterium-based transformation of wheat cultivars and wild relatives with CRISPR-Cas constructs expressing growth regulators GRF-GIF: The KSU wheat transformation facility led by co-PD Trick established the Agrobacterium-based transformation system for wheat. This technological advance combined with the constructs expressing the GRF-GIF growth regulators developed at UC Davis will allow us to conduct direct transformation of adapted cultivars or wild relatives of wheat. This combination of technologies was used by our international collaborator, co-PD Pozniak, to edit adaptive genes in a wild tetraploid ancestor of wheat (see below). Editing the regulatory regions of genes controlling productivity traits NAM-B1 promoter editing: The NAM-B1 allele from wild emmer is not available in the background of transformation-amenable cultivars. To edit the NAM-B1 promoter region, we pursued three strategies. 1) In 2022, we have developed the T0 Bobwhite lines each expressing three multiplexed gRNA constructs (3 gRNA/per construct). These lines are planted for crossing with PI 664549 carrying the wild-type NAM-B1 allele. The progeny of these crosses will be screened for promoter mutations to select lines for developing populations to evaluate the impact of these regulatory mutations on agronomic traits.2) The previously developed T0 Bobwhite lines expressing Cas9 constructs at high levels have been crossed with PI 664549. The BC1F1 generation seeds from these crosses are planted in August 2022 to generate BC2F1 by 2023. The BC2F1 plants will be genotyped using genome-wide skim-seq and Cas9-specific markers to select lines with Cas9 introgression and high proportion of recurrent parent genome. The progeny lines expressing Cas9 will be inoculated with the pool of BSMV-gRNA constructs targeting NAM-B1 promoter. 3) We used the Agrobacterium system with GFR-GIF and multiplexed Cas9-sgRNA constructs (3 sgRNAs/construct) to transform PI 664549. The T0 plants are expected by the end of 2022. Combined, these strategies will ensure that we will generate cis-regulatory variants of the NAM-B1 gene in our project. GRF4 promoter and microRNA396 binding site editing: To create new variants of GRF4 with distinct levels of expression and growth phenotypes, we targeted the GRF4 promoter region (4 sgRNAs designed to regions conserved among homoeologs) and microRNA396 binding sites. Each gRNA targeting these regions was cloned into the BSMV construct, which was used to deliver the gRNAs into cultivar Bobwhite expressing Cas9. A total of 58 M1 lines (17%) derived from BSMV-inoculated plants targeting GRF4 promoter carried mutations in at least one homoeologous copy. Nearly 30% of these M1 lines had mutations in all three homoeologous copies. We identified small scale deletions ranging from single base to tens of base pairs at the sites targeted by each of the 4 sgRNAs. We also identified 5 lines that carry deletions of the promoter regions between the two sgRNA target sites. Thus, we successfully edited the promoter region of GRF4 using BSMV-based sgRNA delivery system. The preliminary visual assessment showed clear differences in the plant height, spike morphology and number of spikelets per spike. Detailed phenotypic evaluation of M2 generation plants derived from these lines will be performed in the greenhouse conditions by February 2023. To edit the GRF4's microRNA396 binding site, we designed two sgRNAs and used both biolistic transformation and BSMV-based sgRNA delivery systems to modify the Bobwhite genome. A total seven M0 BSMV-inoculated plants and five transgenic T0 plants showed evidence of somatic editing (10-40% editing efficiency) at the microRNA396 binding site. The progeny of these plants will be evaluated in 2022-2023 growth season to identify homozygous mutants and assess the effect of mutations of agronomic traits. Additional BSMV-inoculated and transgenic lines are being generated to increase the chance of recovering desired mutations of the microRNA396 site. CRISPR editing of negative regulators of grain number, size and weight The effects of TWG6 gene editing: The TGW6 gene family includes members located on chromosome groups 1, 3 and 7. We have edited a group of four TaTGW6 genes from chromosomes 3A (2 copies), 3B (1 copy) and 3D (1 copy). The 5191-2 line in the background of cultivar Bobwhite had all four TGW6 copies mutated. This line was crossed with Bobwhite to develop F5 population including 200 RILs. These RILs were specifically selected to represent different levels of the TGW6 gene dosage. Phenotyping for yield-related traits in greenhouse conditions showed that increase in the number of edited gene copies from 0 to 3 is associated with increase grain size up to 4% without effect on the grain number. The increase in grain weight appears to be associated with increase in grain length and width. Here, we provide evidence for the positive effect of TGW6 editing on grain weight in wheat. International partnership with the University of Saskatchewan We established partnership with the Canadian wheat genomics and breeding program led by C. Pozniak. In the project, CRISPR-Cas9 system is used for editing genes controlling the domestication and adaptive traits to condition non-adapted genetic lines for deployment in wheat breeding programs. To-date, co-PD Pozniak has constructed a multiplex construct targeting RhtB1, PpdA1, VRN2 and Q genes controlling height, photoperiod response, vernalization requirement and seed threshability in wheat, respectively, and transformed the elite durum wheat cultivar CDC Fortitude to create the TetraOM tester line. Intercrossing of TetraOM and tetraploid emmer wheat accessions will be performed to generate adapted exotic germplasm. In parallel, they have developed a multiplex PE vector pXMCas9-GRF and prime editing gRNA (pegRNA) donor vectors to directly edit the domestication genes in wild emmer. Transformation of emmer wheat Zavitan and TG3487 is currently underway. Additionally, to reduce the reliance on emmer wheat genetic transformation, they are generating Cas9 and nCas9-MMLV expressing elite and emmer wheat lines that could be used for direct delivery of sgRNA/pegRNA using BSMV. In summary, they have established a wheat multiplex gene knockout and prime editing platform that can be used to generate suitable germplasm for wheat breeding.

Publications

Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang W, Yu Z, He F, Bai G, Trick HN, Akhunova A, Akhunov E. Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system. Plant Biotechnol. J. 2022 doi: https://doi.org/10.1111/pbi.13910. Online ahead of print.
Type: Books Status: Published Year Published: 2022 Citation: Wang W, Akhunov E. Application of CRISPR-Cas-based Genome Editing for Precision Breeding in Wheat. In the Wheat Improvement textbook. Eds: M. Reynolds, H. Braun. Springer Cham. 2022. 539-556.
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Akhunov E. Wheat Genomics and Gene Editing. The U.S. Wheat Associates International Staff Training, March 28, 2022, Manhattan, KS.
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E, CRISPR Cas-based gene editing technologies. Genomic Technologies Workshop. Kansas State University, Manhattan, KS, June 6-9, 2022. https://www.k-state.edu/igenomics/workshops/Genomic%20Technologies.html
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stelling N, Wang W, Akhunov E. Development of strategies to improve modern wheat cultivars by adopting genetic diversity from wild relatives. Summer Undergraduate Research Showcase. July 28, 2022, Kansas State University, Manhattan, KS.
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Akhunov E. Applications and regulatory aspects of CRISR-Cas9 editing in wheat. U.S. Wheat Associates World Staff Conference, Maui, Hawaii, Aug. 23, 2022.
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Akhunov E. Update on USDA NIFA/KWC-funded grant on application of gene editing to improve wheat. Kansas Wheat Research Committee. April 20, 2022, Manhattan, KS.
Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Wang W. CRISPR-Cas genome editing technology as a tool for precision breeding in wheat, Graduate seminar class series, Department of Plant Sciences, University of Idaho, Moscow, ID, Oct. 22, 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Wang W. Development and Application of High-Throughput Gene Editing Technology in Wheat, 5th Zhongshan International Young Scientist Symposium, Nanjing Agriculture University, Nanjing, China, May 28, 2022.

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Our target audiences are 1) stakeholders, wheat breeders, farmers, government and industry representatives whose feedback play important role in selecting targets for genome editing project; 2) students and postdoctoral researchers who are interested in application of the genome editing technology in breeding and research; 3) general public whose opinion influences the acceptance of gene editing technology in breeding and development of new crop varieties. Changes/Problems:Special circumstances Due to COVID-19 related situation, lab closure and stay-in-home policy, production of transgenic plants and their phenotypic evaluation was significantly delayed in 2020 and 2021. Due to COVID-19 we also experienced delays with recruiting new graduate students. What opportunities for training and professional development has the project provided?Two postdoctoral researchers (Wei Wang, Zitong Yu) involved into the project have attended the workshop "Barley and Wheat Transformation" organized by the Bill and Melinda Gates Foundation (June 15th, 2021). Three undergraduate students from Kansas State University involved into the research project (Sami Weber, Taiwo Bamidele, and Kynlie Ibarra) received training in phenotyping and genetic characterization of gene editing plants. How have the results been disseminated to communities of interest?Stakeholders, farmers, government and industry representatives: PD Akhunov delivered presentations to industry representatives and stakeholders at the meetings organized by the Kansas Association of Wheat Growers, Kansas Wheat Commission (KWC), and Heartland Plant Innovations Center (HPI) and K-State Research and Extension. We worked with the U.S. Wheat Associates to develop a commentary based on the results of wheat genome editing projects submitted in response to feedback solicitation to "Proposal to Exempt Plants with Additional Modifications Produced Using Genetic Engineering That are Otherwise Achievable by Conventional Breeding": https://www.aphis.usda.gov/aphis/newsroom/stakeholder-info/sa_by_date/sa-2021/sa-07/proposal-to-exempt-plants Wheat researchers, breeders, industry representatives: Project results are also presented to wheat researchers, breeders and industry by preparing a press release in coordination with International Wheat Yield Partnership. The project team presented their discoveries to wheat researchers and breeders at the national and international conferences: 1) BreedWheat Symposium: Advances and Beyond, 2-4 September, 2020, Clermont-Ferrand, France, 2) 2020 ASA-CSSA-SSSA International Annual Meeting, November 9-13, 2020. The results of this gene editing project were also included into the talk presented at the International Wheat Genome Sequencing Consortium Fall 2020 Webinar Series, Oct. 7. The outputs of the project were disseminated to the international wheat community by publishing in the international peer-reviewed journals. The press release about the gene editing project was prepared in collaboration with the IWYP team (IWYP Science Brief No. 17, July 2021; https://iwyp.org/iwyp-science-briefs/). Students and postdoctoral researchers: The KSU team participates in the two monthREEU program run the KSU Department of Plant Pathology by hosting an undergraduate student. What do you plan to do during the next reporting period to accomplish the goals?For CRISPR editing of promoter regions in the NAM-B1, GRF4 and omega-gliadin genes, we have obtained T0 generation plants. In 2021-2022, we will grow and evaluate next-generation of edited plants. The T1 generation of NAM-B1 edited lines is planted and will be grown in the fall of 2021. These plants will be evaluated for the presence of the desired NAM-B1 promoter edits in PI 664549 line. Cross between transgenic Bobwhite carrying multiple gRNAs and PI 664549 carrying the wild-type NAM-B1 gene will be made. The progeny of the latter cross will be screened in 2022 for the presence of the NAM-B1 promoter mutations. Similar strategies will be applied for editing the GRF4 promoter. We will also initiate CRISPR-editing of the Q gene promoter. For editing the omega-gliadin genes, we will be applying a strategy including crossing of Bobwhite lines expressing multiple gRNAs targeting all gene copies with cv. Fielder. In addition, we will test the approach based on inoculation of cv. Fielder expressing Cas9 with BSMV-gRNA constructs. The progeny of this cross carrying the Fielder alleles of omega-gliadins and high expressing Cas9 will be inoculated with the gRNA in BSMV constructs and further screened for mutations in the omega-gliadin genes. In 2021-2022, we will characterize gene editing events in the GASR7, GW7, An1, SPL16, GW2, GS3, and GSE5 genes in the progeny of T1 plants derived from a transgene with the Cas12a-MGE construct subjected to high-temperature treatment. Populations carrying multiple gene editing events will be planted for phenotypic evaluation. In 2021-2022, we will initiate genotypic and phenotypic evaluation of wheat cultivars carrying CRISPR-induced mutations in multiple genes that affect sink-related traits (grain size, weight and number). These wheat cultivars were selected based on the presence of natural alleles at gene loci controlling both source- and sink-related traits and distinct from gene loci subjected to CRISPR editing.

Impacts
What was accomplished under these goals? Editing the regulatory regions of genes controlling productivity traits NAM-B1 promoter editing: While the functional allele of NAM-B1 from wild emmer increases grain protein content (GPC), it is often associated with reduced grain yield (GY). We applied CRISPR for editing the promoter elements of NAM-B1 to obtain new gene variants with reduced negative impact on GY. We designed multiple gRNAs to target the promoter region of NAM-B1. The genome editing strategies were based on: 1) a pool of multiple single-plex gRNAs, and 2) a pool of three multiplexed gRNA constructs, each with three gRNAs. The constructs were used to transform two wheat cultivars: Bobwhite (no NAM-B1) and PI 664549 (carries NAM-B1 from wild emmer). The T0 generation plants have been obtained in both Bobwhite and PI 664549 cultivars for single-plex pool of gRNAs. The next-generation sequencing of these T0 plants revealed that each carry from 4 to 12 gRNAs. The T1 generation was planted and will be grown in the fall of 2021 for further analysis. GRF4 promoter and microRNA396 binding site editing: The GRF4 and Rht-1 have opposing effects on plant growth, nitrogen uptake, and carbon fixation. To create new variants of GRF4 with distinct levels of expression and growth phenotypes, we targeted the GRF4 promoter region and microRNA396 binding sites. We designed multiple gRNAs to target the promoter regions of three homoeologous copies of GRF4 on chromosomes 6A, 6B and D. Two gRNAs were designed for editing the microRNA396 binding site. After testing in the protoplasts, which showed that all designed constructs show acceptable level of editing efficiency, each of the gRNAs was sub-cloned into the Barley Streak Mosaic Virus (BSMV) construct, which will be used for delivering gRNAs into the leaves of wheat line already expressing Cas9 gene. We have demonstrated that the BSMV-based delivery of gRNA is capable of inducing gene edits in the next generation of wheat lines. The Bobwhite line expressing Cas9 at high level was planted to be used for inoculation with the pool of 10 BSMV-gRNA RNAs synthesized using each construct. The GRF4 promoter editing events will be identified in the progeny of BSMV-inoculated lines in the spring of 2022. Engineering a gene affecting end-use quality traits Omega-gliadin gene editing: The functionality of wheat dough is defined by the gluten network formed by the intermolecular disulfide bonds between two cysteine residues. Compared to other gliadin subtypes, omega-gliadins do not contain cysteine residues but are rich in toxic epitopes. The genome editing of omega-gliadins is expected to have low impact on dough quality but substantially reduce its toxicity. There are 18 copies of omega-gliadin genes identified in the genome of cv. Fielder. We designed eight gRNAs targeting omega-gliadin genes across the three genomes. The gRNAs were subcloned into 1) plasmids carrying Cas9 and 2) gamma chain of BSMV construct. The T0 plants transformed with the pool of eight gRNAs in Cas9 plasmids was obtained in spring of 2021 and will be evaluated for gene editing events in the fall of 2021. CRISPR editing of negative regulators of grain number, size and weight TaCKX6-1 and TaCKX6-2 gene editing: The TaCKX6 genes are considered negative regulators of grain number and grain size. There are three copies of TaCKX6 in each wheat genome. We generated knock-out mutations in two gene copies (henceforth, TaCKX6-1 and TaCKX6-2) that have not been edited before. We crossed a triple knockout mutant of TaCKX6-1 with CIMMYT line 3613474. We detected 11% increase in the number of grains per spike in F2 lines carrying 3-6 edited copies of TaCKX6-1 compared to lines carrying 1-3 edited copies. In this experiment, we did not observe significant impact of TaCKX6-1 mutations on grain size and weight. To further confirm the effect of TaCKX6-1 editing, an F3 population of 210 lines has been grown and its phenotypic evaluation is underway. Phenotyping of an F2 population derived from a cross between a double TaCKX6-2 mutant and winter wheat KS12DH0013-41 showed modest 3.6% increase in the number of grains per spike in lines with the A and B genome copies edited compared to wild-type lines. To further confirm the effects of TaCKX6-2 editing, we have grown two F3 and two F4 populations segregating for the TaCKX6-2 mutations in all three genomes and spike- and grain-related trait evaluation is underway. ARF4 gene editing: ARF4 is a negative regulator of grain size and has two similar copies on each of the A, B and D genomes. By characterizing T1 population derived from a line with a knockout mutation in the B genome, we observed 2.5% increase in grain weight. This result was confirmed in T2 population, which showed 3.2% increase in grain weight. To validate the effects of editing of both copies of ARF4, we have developed and harvested F3 generation lines derived from a cross between Bobwhite and T0 plant 7388-1, which carries triple knockout mutations in both copies of ARF4. Collection of grain morphometric trait data is on-going. Broadening the wheat genome editing toolbox Multiplex editing of yield component genes using the Cpf1 (Cas12a) nuclease: The range of editable genome is defined by the ability of CRISPR-based editors to recognize distinct protospacer adjacent motifs (PAMs), and the length and structure of guiding RNAs. We tested Cas12a for multiplex gene editing (MGE) in wheat. The eight-fold improvement in editing efficiency was achieved by using the gRNAs flanked by ribozymes and RNA polymerase II promoter from switchgrass. We showed substantial increase in MGE after subjecting transgenic plants to high-temperature treatment (Wang et al., 2021). Using single MGE construct and high-temperature treatment, we obtained mutations in GASR7, GW7, An1, SPL16, GW2, GS3, and GSE5 genes. The progeny of these mutants is being planted in fall of 2021. Evaluation of prime editing system in wheat: A total of 10 distinct constructs designed for prime editing (PE) have been tested in the wheat protoplasts, including designs with different lengths of primer binding site and different lengths of repair template. These constructs were designed to change G to C in position 71 of the Q gene. The design QT1PE-8-10+3 with the length of prime binding site 8 bp and the length of repair template 10 bp showed the highest editing efficiency (~25%). The PE constructs with similar design are currently being prepared for editing the miRNA binding site of GFR4. International partnership (University of Saskatchewan) We established partnership with the Canadian wheat genomics and breeding program led by C. Pozniak. In the project, CRISPR-Cas9 system is used for editing genes controlling the domestication and plant development traits to condition non-adapted genetic material for easy deployment in the wheat breeding programs. The rapid transient assay to validate the gRNAs and vector elements prior to transformation was developed. We constructed a reporter vector system for co-expression of CRISPR-Cas9 components and fluorescent reporter proteins such as YFP, GFP and mCherry. In this system, expression of YFP, but not the GFP or mCherry, will indicate lack of modification in the target region or indels involving entire codon(s). Preliminary assessment of this system in wheat protoplasts was successful. We initiated development of CRISPR-Cas9 edited Triticum dicoccum and T. durum, targeting edits for domestication and agronomic traits. For the T. dicoccum, guides were designed to optimize flowering time (Ppd), reduced plant height (Rht), and grain threshability genes. For T. durum, we focused on reduced height genes, targeting variants of three know Rht loci. We have successfully developed and have confirmed our primary transgenics. We expect to have seed from these transgenics by September 2021, at which time we will confirm expression and select plants with confirmed mutations for subsequent study.

Publications

Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang W, Tian B, Pan Q, Chen Y, He F, Bai G, Akhunova A, Trick HN, Akhunov E. Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases. Plant Biotechnol. J. 2021 Jul 16. doi: 10.1111/pbi.13669. Online ahead of print.
Type: Book Chapters Status: Awaiting Publication Year Published: 2021 Citation: Wang W, Akhunov E. Application of CRISPR-Cas-based Genome Editing for Precision Breeding in Wheat. In the Wheat Improvement textbook. Eds: M. Reynolds, H. Broun. Springer. 2021 (in press).
Type: Websites Status: Published Year Published: 2021 Citation: Akhunov E. Developing a System for CRISPR-Based Precision Breeding in Wheat. WYP Science Brief No. 17, July 2021 (https://iwyp.org/iwyp-science-briefs/).
Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Akhunov E. Differential chromatin accessibility map as a new resource for studying wheat genome function and genotype-to-trait relationships. International Wheat Genome Sequencing Consortium Fall 2020 Webinar Series, Oct. 7.
Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Akhunov E. Adaptive genomic diversity in wheat and its wild relatives, BreedWheat Symposium: Advances and Beyond, 2-4 September, 2020, Clermont-Ferrand, France.
Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Akhunov E. Integrating eQTL, Genome Editing, Epigenetics and GWAS to Understand the Genetic Control of Phenotypic Traits in Hexaploid Wheat. 2020 ASA-CSSA-SSSA International Annual Meeting, November 9-13, 2020.