Source: KANSAS STATE UNIV submitted to NRP
CRISPR/CAS-BASED EDITING OF GENES AFFECTING YIELD COMPONENT AND QUALITY TRAITS IN HARD WINTER WHEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025623
Grant No.
2021-67013-34174
Cumulative Award Amt.
$490,000.00
Proposal No.
2020-03585
Multistate No.
(N/A)
Project Start Date
Feb 1, 2021
Project End Date
Jan 31, 2025
Grant Year
2021
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
Wheat breeding in the US Great Plains is mostly focused on improving hard winter wheat, which remains one of the major crops in Kansas, with 8-12 million acres planted every year and annual production ranging between 280 and 460 million bushels. Grain yield and grain quality are among the major breeding targets for hard winter wheat. With continued decrease in planted winter wheat acres in Kansas, ability to harvest more grain with increased protein content and bread making quality from smaller area are important factors considered by wheat growers during variety selection. Improving grain yield and quality is a also a main priority for the state's commodity association and milling industry.Recent advances in wheat genetics and genomics helped to better understand the genetic basis of many agronomic traits allowing wheat breeders to accelerate development of new varieties by utilizing this information. The identification of genes that underlie trait variation opened new opportunities for wheat improvement by stacking favourable variants of various genes, which are the part of the biological pathways controlling the same or complementary agronomic traits. The success of this breeding process dependent on the availability of novel variants of genes. While traditionally these variants are introduced into the breeding programs from cultivars from other geographic regions, or close and distant ancestors of wheat, recent advances in genomic technologies provide an opportunity to create novel gene variants using the CRISPR tools. These tools allow researchers to precisely modify the genetic code and create new genes that have potential to improve agronomic traits. Recognizing the future potential of this novel gene editing technologies, Kansas Wheat Commission and the state's wheat growers prioritized this direction of research, and contributed funding to build wheat genome editing capacity at K-State. Our project will build on these resources and expand further the set of tools available for wheat genome editing.We will focus on transferring the CRISPR-induced genes that showed potential to increase grain yield and grain quality into the hard winter wheat cultivars from the Kansas wheat breeding program. We will introduce the CRISPR-induced genes into the wheat cultivars that already have other superior traits, such as improved root system, good bread making quality, high yield, or high grain protein content. By transferring these new variants of genes into the adapted cultivars, we will be able to investigate their interaction with genes that define the superior traits important for wheat production in Kansas, and select optimal combinations of cultivars and CRISPR-induced genes for improving winter wheat. The CRISPR tools previously applied to edit the wheat genome are not effective at performing certain types of manipulations with the genetic code, such as replacement of individual bases in DNA. In our project, we will try to expand the range of DNA alterations possible in the wheat genome by applying novel technology, referred to as "prime editing", to modify genes that have potential to improve wheat productivity.Our project will collaborate with the wheat research and improvement programs at KSU, Kansas Wheat Innovation Center, International Wheat Yield Partnership (IWYP), USDA NIFA Wheat Coordinated Agricultural Project (CAP), and the NIFA IWYP Winter Wheat Breeding Innovation (WWBI) Hub at KSU. Aligning our project priorities with the priorities of these national and international initiatives, we will broaden the project's impact, and facilitate incorporation of the project outcomes into the breeding pipelines of private and public breeding programs. We will work on broadening the training opportunities in genome editing technologies for graduate students, researchers and breeders. Our efforts will contribute to the Kansas wheat breeding program's mission to develop strategies for delivering high-yielding wheat varieties with improved quality to Kansas framers, and address one of the key priorities of the NIFA USDA program to improve crop productivity and quality in the local U.S. farming systems.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011540108180%
2011549108020%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other; 1540 - Hard red winter wheat;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Overall goal: Evaluate the effects of CRISPR-Cas-induced diversity on the targeted traits assessed in the winter wheat germplasm under field conditions, and investigate interaction between the engineered alleles and productivity and grain quality QTL existing in winter wheat germplasm. Devise better strategies for incorporating the CRISPR-induced alleles into the hard winter wheat breeding process.The specific objectives of the project:1) CRISPR-assisted transfer of validated gene variants affecting yield component traits into the hard winter wheat germplasm.2) Apply prime editing technology for creating base substitutions in the coding regions of genes affecting wheat productivity traits.3) Evaluate the effects of gene editing on yield and grain quality traits in hard winter wheat germplasm.4) Broadening training opportunities in genome editing technologies for graduate students and postdoctoral researchers.
Project Methods
Selection of recipient hard winter wheat germplasm carrying complementary traitsThe recipient lines for genome editing will be selected among the hard winter wheat lines developed by co-PD A. Fritz, or lines broadly used in the Great Plains' breeding programs. Many of these lines have been also selected as recipient germplasm for trait conversion in the NIFA IWYP WWBI Hub based on applicability to US germplasm, accessibility in terms of IP to use as breeding parents (as defined by the "breeder's exemption" of the US PVP and UPOV rules), commercial interest, crossability to other germplasm, quality characteristics, phenology, and also availability of lines carrying introgression of male sterility gene Ms3.Among selected germplasm, we will have lines with introgression from closest wild relatives, Aegilops tauschii and Triticum dicoccoides. We will focus on lines carrying introgression of wild alleles associated with variation in productivity and nutritional quality traits.All lines in the project have been re-sequenced using an exome capture assay providing an opportunity to compare the genotypes of each target line with lines in the public databases so haplotypes in the genomic regions carrying important agronomic genes can be inferred and for which genetic background selection can be conducted.Transfer of gene editing events into the recipient germplasmTo ensure successful development of winter wheat germplasm with the CRISPR-edited variants of genes, we will apply several strategies for introgression of novel engineered alleles. First, we will focus on using the standard marker-assisted background selection strategy based on the KASP markers developed for foreground selection of CRISPR-induced alleles. Second, we will use the previously tested strategy based on crossing cv. Bobwhite expressing Cas9 transgene at high levels with a recipient germplasm. In the F1 hybrids, this approach allows for the efficient genome editing of the recipient's gene copies leading to non-Mendelian segregation of edited vs. non-edited alleles in the progeny. The two rounds of backcrossing to a recipient parent combined with foreground marker-assisted selection will be used to recover the recurrent parent's genome. The third strategy will be based on the HI-Edit technology, which relies on delivering the sgRNA-Cas9 ribonucleoprotein complexes into the wheat embryo using maize pollen. Independence of this technology from the genotype of the recipient plant provides opportunity for editing a diverse range of germplasm.Testing Prime Editing (PE) technologyWe will apply the recently developed PE technology for editing genes (TaSPL14, TaSPL16 and Q), which have potential to improve wheat productivity traits. Due to the low efficiency of HDR-based CRISPR editing in wheat, so far we have been unsuccessful in making precise sequence insertions or replacements in the wheat genome. In all three selected genes, alleles associated with increased productivity carry mutations at the microRNA binding sites that reduce transcripts' affinity to matching microRNAs. To create beneficial alleles of these genes in the wheat genome, we will perform base replacements at the microRNA binding sites using PE and pegRNAs.The spacer sequence parts of the pegRNA for guiding prime editing machinery to targets will be designed using the sgRNA Scorer 2.0 and CRISPR-DT programs that allow for including the custom target sequences for guide RNA design. The spacer sequences will be compared to the cv. Chinese Spring reference genome to select unique spacers and reduce off-target editing. All PE constructs will be tested for editing efficiency by transient expression in the wheat protoplasts. The frequency of editing events in the wheat genome will be estimated by the highly multiplexed next-generation sequencing (NGS) of the targeted regions.Development of the transgenic wheat lines using the PE constructsThree PE constructs will be co-bombarded with the bar gene construct that is used for transformant selection in the growth media with glufosinate. Each glufosinate-resistant tiller of a transgenic plant will be tested for the presence of PE construct by PCR. The expression of nCas9/RT genes in the PE-positive tillers will be analyzed by quantitative RT-PCR. Base substitutions at the microRNA binding sites of targeted genes will be identified by the NGS of barcoded PCR amplicons.Phenotypic evaluationDepending on timeframe required to develop the CRISPR-edited wheat lines and the amount of seeds required, phenotypic evaluation will be performed in the field and/or greenhouse conditions. Plants produced using prime editing and HI-Edit technology will be phenotyped at both T1 and T2 generations in the greenhouse conditions. The BC2F2:3 lines with CRISPR-edited genes in the hard winter wheat background will be phenotyped in both greenhouse and field conditions. We will also use three adapted hard winter wheat cultivars (Smith's Gold, Danby and KanMark) as checks to compare with the CRISPR-edited wheat lines.Standard agronomic performance traits including timing of stem elongation, heading date, time to anthesis, date of physiological maturity, and plant height will be measured. Wheat productivity will be assessed based on the estimates of yield, test weight, thousand grain weight, the number of fertile tillers/spikes per plant or per unit area, average number of grains per spike, and average grain weight. To assess the source-sink trait relationships in the CRISPR-edited lines, we will measure the harvest index.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. 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.All generated data will be made available to wheat breeders and researchers through the Wheat CAP's T3 database. Educational activitiesOur project in coordination with the KSU Integrated Genomics Facility (IGF) will develop educational modules for the yearly summer workshop (2 credit course) to train postdoctoral researchers and PhD students in the application of genome editing technologies for crop improvement. The IGF personnel will conduct on-line course evaluation to assess the outcome of training. Our team is also involved in training activities of the Wheat CAP (two co-PDs from this project serve as co-PDs of Wheat CAP); this connection will allow for incorporating genome editing training modules into the Wheat CAP's educational activities that provide training to a larger cohort of graduate students.

Progress 02/01/21 to 01/31/25

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:Due to COVID-19 related situation, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021 and 2022, which affected the progress with developing introgression populations in winter wheat background. As result, field-based evaluation of the developed germplasm with introgression of gene edited alleles will be finalized outside the timeframe of this current project. The prime-editing technology initially proposed for one of the gene editing objectives showed extremely low editing efficiencyin wheat making it unsuitable for the project's objectives. As replacement for this technology, we have developed virus-based gene editing system that provides opportunities for high throughput genome editing without need to transform wheat with the gene editing constructs. What opportunities for training and professional development has the project provided?Two postdoctoral researchers (Wei Wang, Zitong Yu) attended the workshop "Barley and Wheat Transformation" organized by the Bill and Melinda Gates Foundation (June 15th, 2021). Educational activities: 1) 2-month summer training (2022) for undergraduate student Nick Stelling from ISU who joined PD Akhunov lab for the KSU REU project (https://www.plantpath.k-state.edu/academics/undergraduate-programs/reeu/projects.html). Nick Stelling joined our lab in 2023 as a graduate student to pursue research in wheat breeding and genetics. 2) In June 2022, postdoctoral researchers from Akhunov Lab delivered lectures on genomics and applications of genome editing to graduate students and postdoctoral researchers enrolled into the annual Genomics Technologies workshop organized by the KSU Integrated Genomics Facility. A graduate student (Nick Stelling) gained experience in scientific communication by presenting the results of his research to scientific community. He has made three poster presentations at ASA, CSSA, SSSA International Annual Meeting (Oct 29-Nov 1, 2023. St. Louis, MO, USA), National Association of Plant Breeders 2023 Annual Meeting (Clemson University. Greenville, SC, July 16-20, 2023), and Plant and Animal Genome 30 Conference (January 12 - January 17, 2024. San Diego, CA, USA). He also delivered two oral presentations about his research projects at the Wheat CAP graduate student workshop (San Diego, Jan. 12, 2024) and Student proposal seminar series at the Department of Plant Pathology, KSU Manhattan, KS. April 3, 2024. 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). For her work on editing the gliadin genes that helped to develop a wheat line with the reduced immunoreactivity, postdoctoral researcher Z. Yu became a recipient of 2024 Jeanie Borlaug Laube Women in Triticum (WIT) Award: https://bgri.cornell.edu/bgri-announces-2024-wit-awardees/?fbclid=IwAR1pxUOyB_nGuXLEv50EXvYwcVB_fpg9rVYyjn8DJeJxz7PxTemnO8PAV5E . The WIT award is named after Jeanie Borlaug Laube, who is a strong advocate for women in agriculture and also daughter of Nobel laureate Dr. Norman Borlaug. The award recognizes talent and dedication among early-career scientists in the field of wheat research. In June 2024, postdoctoral researchers and graduate students from Akhunov Lab delivered lectures on genomics and applications of genome editing to graduate students and postdoctoral researchers enrolled into the annual Genomics Technologies workshop organized by the KSU Integrated Genomics Facility. 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. PD Akhunov delivered presentations at the meetings with industry: 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 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. 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 PD Akhunov delivered five presentations at the international meetings: 1) FROM SEED TO PASTA IV, Bologna - Italy, 26-29 October 2022; 2) 6th International Wheat Yield Partnership Program Conference, 4 - 5 October, 2022; 3) Wheat Associates World Staff Conference, August 21 to 25, 2022 in Maui, HI, USA. 3) USDA NIFA Project Director Meeting at the National Association of Plant Breeders. 2023 Annual Meeting. Clemson University. Greenville, SC; 4) 7th International Wheat Yield Partnership Program Conference, 27 & 28 September 2023; 5) Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA. PD Akhunov disseminated the results of research funded by this project to general audience by participating in a podcast and giving an interview to public media: 1) Recording of podcast with Aaron Harris: Wheat's On Your Mind podcast. www.wheatsonyourmind.com. The podcast discussed the usage of wheat genome editing to reduce gluten. This podcast follows the research published in the Plant Biotechnology Journal (Yu et al., 2023). 2) TV interview to WIBW channel follows the same gene edited wheat with reduced gluten story: https://www.wibw.com/2023/12/20/k-state-researchers-lower-gluten-content-maintain-dough-quality-wheat-bread/ A graduate student (Nick Stelling) made three poster presentations at the international meetings: ASA, CSSA, SSSA International Annual Meeting (Oct 29-Nov 1, 2023. St. Louis, MO, USA), National Association of Plant Breeders 2023 Annual Meeting (Clemson University. Greenville, SC, July 16-20. 2023), and Plant and Animal Genome 31 Conference (January 12 - January 17, 2024. San Diego, CA, USA). He also delivered two oral presentations about his research projects at the Wheat CAP graduate student workshop and at Student proposal seminar series at Plant Pathology, KSU Manhattan, KS. April 3, 2024. Postdoctoral researcher Z. Yu presented her work at the Plant and Animal Genome 31 (January 12 - January 17, 2024. San Diego, CA, USA) and PAG32 (January, 2025. San Diego, CA, USA). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Novel transformation-free editing approach using the BSMV-based delivery of gRNAs: We have developed a novel, transformation-free method for editing the wheat genome. This approach relies on the inoculation of Cas9-expressing wheat lines with Barley Stripe Mosaic Virus (BSMV) RNA carrying guide RNAs (gRNAs) embedded in the γ (gamma) chain of the viral genome. We demonstrated that multiplex genome editing can be achieved using this method in the cultivars Fielder and Bobwhite, both transformed with the Cas9 gene. The method was initially applied to generate heritable, precise deletions in the promoter region of the AP2 transcription factor encoded by the Q gene and to perform multiplexed editing of agronomic genes (TaGW2, TaUPL3, TaGW7) associated with yield component traits. Introgression of the Cas9 locus into winter and spring wheat cultivars enabled efficient editing in adapted germplasm. Using this strategy, we edited TaGW7 in both spring and winter wheat lines from CIMMYT and Kansas breeding programs. This approach is now being used to generate edits directly in adapted germplasm selected for this project. Given that BSMV-sgRNAs can be designed to target hundreds of loci and the inoculation procedure is simple, Cas9-introgressed lines now serve as a platform for high-throughput editing of multiple genes in wheat to improve its agronomic performance. The results of this study have been published in Plant Biotechnology Journal. (Wang et al., 2022). New edited alleles of TaGS3 for increasing grain size and weight in wheat: In rice, loss-of-function (LOF) mutations in GS3 increase grain size, weight, and yield. However, the effects of GS3 LOF mutations on these traits in wheat remained unknown. In our study, we characterized for the first time the effects of TaGS3 editing on yield component traits in wheat. Contrary to findings in rice, knockout mutants with all TaGS3 copies edited exhibited a 7.5% reduction in grain weight (p = 1.5E-4) and a 4.4% reduction in grain length (p = 1.7E-8). The greatest increases in grain weight and area were observed in mutants carrying an intermediate number of LOF alleles. Two double mutants, one with TaGS3 knockouts on chromosomes 7A and 4A, and the other on chromosomes 7A and 7D, showed 7.9% (p = 6.4E-3) and 7.2% (p = 2.4E-3) increases in grain weight, respectively. The single-locus TaGS3-4A mutant exhibited a 9.5% increase in grain weight (p = 1.1E-3). Grain size traits in the edited lines followed a pattern similar to that of grain weight. TaGS3 editing had no significant impact on grain number per head. The novel genome-edited germplasm developed in this study offers a promising resource for increasing grain weight in wheat improvement programs. The results of this work have been published in Theoretical and Applied Genetics (Wang et al., 2025). Editing of gluten-encoding genes to reduce immunogenic response of wheat lines and improve bread-making quality: Gluten sensitivity caused by peptides produced during digestion of wheat products could be reduced by removing genes encoding gluten proteins. However, attempts to reduce gluten protein content by RNAi silencing and gene editing often led to a reduction in end-use quality characteristics of wheat flour. We have developed a gene-edited line, #387-3-6, which had all omega-gliadin gene copies and more than half of the gamma-gliadin genes edited or deleted. Using G12 and R5 antibodies, we showed a 47-fold reduction in the immunogenic response of protein extracts prepared from line #387-3-6 compared to those from the non-edited cultivar Fielder. Compared to non-edited lines, we observed significant increases in the ratio of glutenins to gliadins (from 1.29 to 1.53), polymeric proteins to monomeric proteins (from 1.10 to 1.22), and in the percentage of glutenin macropolymer (from 51.6% to 53.2%). These results suggest that reduction of ω-gliadins and γ-gliadins facilitates gluten polymerization, which positively correlates with dough quality parameters. The results of dough quality testing conducted using the Mixograph and Farinograph confirmed these conclusions. These results indicate that the developed line #387-3-6 could be used in breeding programs to reduce immunoreactivity and improve end-use quality traits in wheat. The results of this study have been published in Plant Biotechnology Journal (Yu et al., 2023). The novel low-allergenecity alleles of gluten genes developed in this project are now being introgressed into winter wheat cultivars to develop low-coeliac wheat varieties. Editing regulatory regions of genes affecting grain yield: Increase in the expression of SPL14 affects plant architecture and increases yield. We applied the BSMV-based genome editing system developed by our group to create in-frame mutations in the miR156-binding site that suppresses TaSPL14 expression in wheat. By screening multiple M1 generation plants, we identified lines with 1-bp, 3-bp, 9-bp, and 15-bp deletions at the target sites. The 3-bp and 15-bp deletion mutants did not produce viable progeny and have been excluded from analyses. Consistent with the functional importance of TaSPL14 in controlling processes affecting yield component traits, the 1-bp frameshift deletion mutant, which likely had a negative impact on protein function, showed significant reductions in grain weight (-13%) and size (-8%). The population segregating for the presence/absence of the 9-bp in-frame deletion is currently being grown to evaluate the effect of this mutation on yield component traits in the summer of 2025. We expect that the in-frame deletion at the miR156 site should not have a negative impact on SPL14 function and should result in increased expression of this gene and a positive impact on yield component traits. Introgression of edited alleles into adapted wheat germplasm: The heterogeneous inbred families (HIFs) with the introgression of the edited TaGW2, TaCKX6-1, TaCKX6-2, TaGW7, and TaARF4 gene alleles have been developed in the backgrounds of spring wheat cultivars IDO676 and Tekoa, and winter wheat cultivars Bob Dole, OK13625, KS090387K-20, KS080093K-18, and OK16D101089. The homozygous lines with and without the edited alleles derived from these HIFs are undergoing greenhouse and field evaluation in replicated experiments. The preliminary results suggest that introgression of TaGW2 into adapted backgrounds increases grain weight by 5-11%, consistent with the previously described effects of TaGW2 on yield component traits. Using the virus-based system of gRNA, we have also performed direct editing of TaGW7 in the winter wheat cultivar KS080093K-18. The analyses of TaGW7-edited lines showed up to a 3-4% increase in grain weight compared to non-edited controls. Introgression of TaGW7 alleles from wild emmer into wheat positively affects grain weight: Our earlier study showed that the editing of TaGW7 results in shorter and wider grains and increased grain weight (Wang et al., 2019). Diversity analyses confirmed that TaGW7 is a potential target of domestication. By analyzing the natural diversity at GW7-A1 in wild and domesticated wheat, we have shown that domesticated forms carry only the low-expressing allele of TaGW7 with a 1.9-kb insertion in the promoter. The high-expressing allele of GW7-A1 from wild wheat lacks this 1.9-kb insertion. Introgression of the high-expressing allele from wild emmer into the Bobwhite cultivar resulted in a 3.8% increase in grain length (p-value = 1.8E-12) and a 5.7% increase in thousand grain weight (p-value = 2.9E-3). These results suggest that introgression of the high-expressing TaGW7 allele from wild wheat has the potential to improve yield component traits in wheat.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Wang W, 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. Theor. Applied Genet. 2025, 138, 38.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Yu Z, Yunusbaev U, Fritz A, Tilley M, Akhunova A, Trick Harold, Akhunov E. CRISPR-based editing of the ?- and ?-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality, Plant Biotechnology Journal, 2024, 22 (4), 892-903.
  • Type: Peer Reviewed 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, 20(12):2332-2341.
  • Type: Conference Papers and Presentations Status: Published 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. 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: Published 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.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. Leveraging modern genomic resources to identify adaptive variants and optimize breeding strategies in wheat. FROM SEED TO PASTA IV, Bologna - Italy, 26-29 October 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. CROP BREEDING INNOVATION HUB: NIFA IWYP Winter Wheat Breeding Innovation Hub (WWBI) at Kansas State University. 6th International Wheat Yield Partnership Program Conference, 4 - 5 October, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. Potential Application of High-Throughput Gene Editing in Wheat Breeding. 6th International Wheat Yield Partnership Program Conference, 4 & 5 October 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. CRISPR/Cas-based editing of wheat, U.S. Wheat Associates World Staff Conference, August 21 to 25, 2022 in Maui, HI, USA.
  • Type: Other Status: Other 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 and 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.
  • Type: Other Status: Other Year Published: 2023 Citation: Akhunov E. Overview of the WGRC IUCRC project and gene editing research at KSU. Bay State Milling Meeting, KWIC, Manhattan, KS, May 31, 2023.
  • Type: Other Status: Other Year Published: 2023 Citation: E. Akhunov. Application of multiplex genome editing strategies for engineering regulatory regions and complex gene loci in wheat. Meeting with the Bimbo Bakeries technical group. Kansas Wheat Innovation Center, Manhattan, KS. April 19, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stelling N, Wang W, Akhunov E. The Potential Effects of GW7 on Grain Dimensions and Plant Height in Wheat Domestication. ASA, CSSA, SSSA International Annual Meeting. Oct 29-Nov 1, 2023. St. Louis, MO, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stelling N, Wang W, Akhunov E. TaGW7 is a Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. National Association of Plant Breeders 2023 Annual Meeting. Clemson University. Greenville, SC, July 16-20. 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. PE0388 TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Plant and Animal Genome 30 Conference. January 12 - January 17, 2024. San Diego, CA, USA.
  • Type: Websites Status: Published Year Published: 2023 Citation: Recording of podcase with Aaron Harris: Wheats On Your Mind podcast. www.wheatsonyourmind.com. The podcast discussed the usage of wheat genome editing to reduce gluten. This podcast follows the research project published in the Plant Biotechnology Journal (Yu et al., 2024).
  • Type: Websites Status: Published Year Published: 2023 Citation: TV interview to WIBW channel follows the work that resulted in developing a gene edited wheat with reduced gluten: https://www.wibw.com/2023/12/20/k-state-researchers-lower-gluten-content-maintain-dough-quality-wheat-bread/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Wheat CAP workshop poster session, January 14, 2024. San Diego, CA, USA.
  • Type: Other Status: Other Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Wheat CAP graduate student workshop, oral presentation, January 12, 2024. San Diego, CA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yu Z, Akhunova A, Trick H, Akhunov E. Application of Multiplexed Gene Editing Strategies for Editing the Regulatory Regions of the Wheat Genome. Plant and Animal Genome 31, January 12 - January 17, 2024. San Diego, CA, USA.


Progress 02/01/23 to 01/31/24

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:Due to COVID-19 related situation, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021 and 2022, which affected the progress with developing introgression populations in winter wheat background. What opportunities for training and professional development has the project provided? A graduate student (Nick Stelling) gained experience in scientific communication by presenting the results of his research to scientific community. He has made three poster presentations at ASA, CSSA, SSSA International Annual Meeting (Oct 29-Nov 1, 2023. St. Louis, MO, USA), National Association of Plant Breeders 2023 Annual Meeting (Clemson University. Greenville, SC, July 16-20, 2023), and Plant and Animal Genome 30 Conference (January 12 - January 17, 2024. San Diego, CA, USA). He also delivered two oral presentations about his research projects at the Wheat CAP graduate student workshop (San Diego, Jan. 12, 2024) and Student proposal seminar series at the Department of Plant Pathology, KSU Manhattan, KS. April 3, 2024. 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). For her work on editing the gliadin genes that helped to develop a wheat line with the reduced immunoreactivity, postdoctoral researcher Z. Yu became a recipient of 2024 Jeanie Borlaug Laube Women in Triticum (WIT) Award: https://bgri.cornell.edu/bgri-announces-2024-wit-awardees/?fbclid=IwAR1pxUOyB_nGuXLEv50EXvYwcVB_fpg9rVYyjn8DJeJxz7PxTemnO8PAV5E . The WIT award is named after Jeanie Borlaug Laube, who is a strong advocate for women in agriculture and also daughter of Nobel laureate Dr. Norman Borlaug. The award recognizes talent and dedication among early-career scientists in the field of wheat research. In June 2023, postdoctoral researchers and graduate students from Akhunov Lab delivered lectures on genomics and applications of genome editing to graduate students and postdoctoral researchers enrolled into the annual Genomics Technologies workshop organized by the KSU Integrated Genomics Facility. How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered two presentations at the meetings with industry: 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 outputs of the project are disseminated to the international wheat community by publishing in an international peer-reviewed journal. Students and postdoctoral researchers: PD Akhunov delivered three presentations at the international meetings: 1) USDA NIFA Project Director Meeting at the National Association of Plant Breeders. 2023 Annual Meeting. Clemson University. Greenville, SC; 2) 7th International Wheat Yield Partnership Program Conference, 27 & 28 September 2023; 3) Plant and Animal Genome 30 Conference. January 13-18, 2023. San Diego, CA, USA. PD Akhunov disseminated the results of research funded by this project to general audience by participating in a podcast and giving an interview to public media: 1) Recording of podcast with Aaron Harris: Wheat's On Your Mind podcast. www.wheatsonyourmind.com. The podcast discussed the usage of wheat genome editing to reduce gluten. This podcast follows the research published in the Plant Biotechnology Journal (Yu et al., 2023). 2) TV interview to WIBW channel follows the same gene edited wheat with reduced gluten story: https://www.wibw.com/2023/12/20/k-state-researchers-lower-gluten-content-maintain-dough-quality-wheat-bread/ A graduate student (Nick Stelling) made three poster presentations at the international meetings: ASA, CSSA, SSSA International Annual Meeting (Oct 29-Nov 1, 2023. St. Louis, MO, USA), National Association of Plant Breeders 2023 Annual Meeting (Clemson University. Greenville, SC, July 16-20. 2023), and Plant and Animal Genome 31 Conference (January 12 - January 17, 2024. San Diego, CA, USA). He also delivered two oral presentations about his research projects at the Wheat CAP graduate student workshop and at Student proposal seminar series at Plant Pathology, KSU Manhattan, KS. April 3, 2024. Postdoctoral researcher Z. Yu presented her work at the Plant and Animal Genome 31 (January 12 - January 17, 2024. San Diego, CA, USA). What do you plan to do during the next reporting period to accomplish the goals?Identify homozygous BC3F2 generation of adapted wheat lines for field testing with the introgression of genes encoding TaGW2, TaCKX6-1, TaCKX6-2, TaGW7 and TaARF4. Isolate homozygous gene edited lines with mutations in the NAM-B1 promoter and SPL14. Assess the effects of these mutations on gene expression, grain protein content, plant architecture and yield components traits (grain size, grain weight and number). Assess effects of mutations in the TaGW7 and Btr1 genes in the background of wild emmer on domestication traits (rachis brittleness, grain dimensions). Phenotypic evaluation of developed lines for relevant agronomic traits, including grain size and number, grain weight, plant height, heading date, protein quantity and quality, etc.

Impacts
What was accomplished under these goals? Impact of COVID on the project: Due to COVID-19, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021 and 2022. These delays resulted in slower progress with the introgression of the edited alleles into adapted backgrounds. Introgression of edited alleles into adapted winter wheat germplasm: We continue making crosses and marker-based selection for transferring edited variants of TaGW2, TaCKX6-1, TaCKX6-2 and TaGW2, TaGW7 and TaARF4 genes. This spring, BC2F2 crosses have been analyzed between donors of these edited alleles and recipients OK16D101089, Bob Dole, OK13625, KS090387K-20, SF Fortify, Tekoa and IDO676. The lines with and without edited alleles of yield-related genes have been identified for planting in the fall of 2024 for field-based evaluation. Introgression of TaGW7 alleles from wild emmer into wheat positively affects grain weight: Our earlier study showed that the knock-out of TaGW7 results in shorter and wider grains and increased grain weight (Wang et al., 2019). The selective sweep analyses showed that TaGW7 is a potential target of domestication. The GW7-A1 alleles in hexaploid wheat and domesticated emmer wheat have a 1.9 kb insertion in the promoter region, which results in decreased expression and present at intermediate frequency in wild emmer. In wild emmer, we have identified the allele of GW7-A1 without 1.9-kb insertion which shows 2-fold higher expression level than the allele without insertion. To investigate the phenotypic effects of the 1.9 kb insertion, the TaGW7-A1 allele from wheat cultivar Bobwhite was introgressed into wild emmer, and the high-expressing alleles of GW7 from the A and B genomes of wild emmer were introgressed into the Bobwhite cultivar. The introgression from Bobwhite into wild emmer did not have significant effects on grain size and weight. However, Bobwhite lines with the introgression of the high-expressing allele from wild emmer showed 3.8% increase in grain length (p-value = 1.8e-12) and 5.7% increase in thousand grain weight (p-value = 2.9e-3). These results suggest that TaGW7 is one of the genes targeted by domestication selection and that re-introduction of the high-expressing TaGW7 allele from wild emmer into wheat has potential to improve grain size and weight traits in wheat. New edited alleles of TaGS3 for increasing grain size and weight in wheat: Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding G-gamma subunit of the multimeric G protein complex, increase grain size and weight in rice. While association between allelic variation in GS3 homologs of wheat and grain weight/size was detected previously, the effects of LOF alleles on these traits remained unknown. We used genome editing to create the TaGS3 mutant lines with the LOF homeo-allele dosage variation. The manuscript presenting the results of this work have been submitted to a journal and published as pre-print in BioRxiv (Wang et al., 2024). The analyses of variation in gran length (GL), grain width (GW), grain area (GA), grain number per spike, and thousand grain weight (TGW) were performed in two advanced generation populations derived from independently developed transgenic plants 4906-1 and C538-1. Contrary to results obtained for rice, editing of all three TaGS3 copies results in significant decrease in GL, GW, GA and TGW, without affecting the number of grains per spike. The knockouts in all TaGS3 copies led to 7.5% reduction in TGW (p = 1.5 x 10-4), 4.4% reduction in GL (p = 1.7 x 10-8). Compared to wild type, the highest increase in grain weight and area was observed in mutants with the intermediate dosage of the LOF alleles, indicating that suppressive effects of TaGS3 on grain size and weight in wheat are dosage-dependent and non-additive. In two double-mutants of TaGS3, with genotypes aabbDD (TaGS3 knockouts on chromosomes 7A and 4A) and aaBBdd (TaGS3 knockouts on chromosomes 7A and 7D), we observed 7.9% (p-value = 6.4 x 10-3) and 7.2% (p-value = 2.4 x 10-3) increase in TWG, respectively, compared to wild type line. The only single-locus mutant AAbbDD that showed 9.5% increase in TGW (p-value = 1.1 x 10-3) was located in the TaGS3-4A homoeolog, which is also the homoeolog that is expressed at the highest level in developing grain. Similar relationships observed between the genotypes of mutated TaGS3 and TGW were also observed for GW and GA traits. The TaGS3 gene editing had no detectable effects on grain number per spike. Our results indicate that 1) fixation of three homoeologous copies of TaGS3 for LOF alleles results in GL, TGW and GA reduction, 2) optimal expression of GL, TGW and GA traits in edited lines is observed in lines with the intermediate dosage of the LOF TaGS3 alleles, and 3) certain combination of the LOF homeo-alleles have significant positive effect on GL, TGW and GA traits suggestive of epistatic interaction between genomes. Overall, our work provides further insights into the complex genetic control of grain dimension and weight traits by the TaGS3 homoeologous loci in wheat. We showed that TaGS3 acts not only as a negative regulator of grain length and weight in wheat. The presence of certain number of the functional TaGS3 gene copies appears to be critical for optimal trait expression. Using CRISPR-Cas9 editing, we have developed the LOF homeo-alleles for TaGS3 and identified the single- and two-locus combinations of these alleles that have potential to increase grain length and weight. The newly developed LOF alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that could be used for increasing grain weight in wheat. Editing regulatory regions of genes affecting grain yield and quality: Increase in the expression of SPL14 affects plant architecture and increases yield. SPL14 expression is repressed by miR156 that binds to a region conserved across all three homoeologs of TaSPL14 in wheat. Our objective was to edit the miR156 binding site and increase TaSPL14 expression through creating in-frame mutations using BSMV-based genome editing approach developed by our group. Two gRNAs were designed to target the miR156 binding site were subcloned into the BSMV construct. Out of all plants inoculated with BSMV, 11 showed somatic editing efficiency >10%. The M1 generation progeny of four M0 plants was screened for the presence of in-frame edits within the miR156 binding site using Illumina MiSeq platform. Among 388 M1 plants, there were two plants with 15-bp and 3-bp deletions resulting in in-frame mutations in coding sequence of TaSPL14 homoeologs from the D and B genomes, respectively. The deleted fragments overlapped with the miR156 binding site (5'-gtgctctctctcttctgtca-3'). The 15-bp deletion results in removal of 9-bp fragment within the miR156 binding site and 6-bp fragment within the CDS of TaSPL14. The 3-bp deletion results in the removal of 3-bp fragment within the miR156 binding site. This 3-bp deletion is expected to increase the expression of TaSPL14 and have minor effect on gene function. The seeds harvested from these two mutants have been planted for phenotypic evaluation in summer of 2024.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Wang W, 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: Journal Articles Status: Published Year Published: 2024 Citation: Yu Z, Yunusbaev U, Fritz A, Tilley M, Akhunova A, Trick Harold, Akhunov E. CRISPR-based editing of the ?- and ?-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality, Plant Biotechnology Journal, 2024, 22 (4), 892-903.
  • 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: 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.
  • Type: Other Status: Other Year Published: 2023 Citation: Akhunov E. Overview of the WGRC IUCRC project and gene editing research at KSU. Bay State Milling Meeting, KWIC, Manhattan, KS, May 31, 2023.
  • Type: Other Status: Other Year Published: 2023 Citation: E. Akhunov. Application of multiplex genome editing strategies for engineering regulatory regions and complex gene loci in wheat. Meeting with the Bimbo Bakeries technical group. Kansas Wheat Innovation Center, Manhattan, KS. April 19, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stelling N, Wang W, Akhunov E. The Potential Effects of GW7 on Grain Dimensions and Plant Height in Wheat Domestication. ASA, CSSA, SSSA International Annual Meeting. Oct 29-Nov 1, 2023. St. Louis, MO, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 4. 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: Stelling N, Wang W, Akhunov E. TaGW7 is a Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. National Association of Plant Breeders 2023 Annual Meeting. Clemson University. Greenville, SC, July 16-20. 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. PE0388 TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Plant and Animal Genome 30 Conference. January 12 - January 17, 2024. San Diego, CA, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Wheat CAP workshop poster session, January 14, 2024. San Diego, CA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Stelling N., Wang W., Akhunov E. TaGW7 Is the Target of Domestication Selection for Grain Dimensions and Plant Height in Wheat. Wheat CAP graduate student workshop, oral presentation, January 12, 2024. San Diego, CA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yu Z, Akhunova A, Trick H, Akhunov E. Application of Multiplexed Gene Editing Strategies for Editing the Regulatory Regions of the Wheat Genome. Plant and Animal Genome 31, January 12 - January 17, 2024. San Diego, CA, USA.


Progress 02/01/22 to 01/31/23

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?Educational activities: 1) 2-month summer training (2022) for undergraduate student Nick Stelling from ISU who joined PD Akhunov lab for the KSU REU project (https://www.plantpath.k-state.edu/academics/undergraduate-programs/reeu/projects.html). Nick Stelling joined our lab in 2023 as a graduate student to pursue research in wheat breeding and genetics. 2) In June 2022, postdoctoral researchers from Akhunov Lab delivered lectures on genomics and applications of genome editing to graduate students and postdoctoral researchers enrolled into the annual Genomics Technologies workshop organized by the KSU Integrated Genomics Facility. How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered two 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. 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 hosted undergraduate students from the two-month REEU program run by the KSU Department of Plant Pathology. PD Akhunov delivered four presentations at three international meetings: 1) FROM SEED TO PASTA IV, Bologna - Italy, 26-29 October 2022; 2) 6th International Wheat Yield Partnership Program Conference, 4 - 5 October, 2022; 3) Wheat Associates World Staff Conference, August 21 to 25, 2022 in Maui, HI, USA. What do you plan to do during the next reporting period to accomplish the goals?Identify homozygous mutants in adapted wheat lines for field testing with the introgression of edited variants of TaGW2, TaCKX6-1, TaCKX6-2, TaGW7 and TaARF4. Isolate homozygous gene edited lines with mutations in the NAM-B1 promoter and SPL14. Assess the effects of these mutations on gene expression, grain protein content, plant architecture and yield components traits (grain size, grain weight and number). Phenotypic evaluation of developed lines for relevant agronomic traits, including grain size and number, grain weight, plant height, heading date, protein quantity and quality, etc.

Impacts
What was accomplished under these goals? Impact of COVID on the project: Due to COVID-19 related situation, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021 and 2022. Introgression of edited alleles into adapted winter wheat germplasm: We continue making crosses and marker-based selection for transferring edited variants of TaGW2, TaCKX6-1, TaCKX6-2 and TaGW2, TaGW7 and TaARF4 genes. This spring, we have finalized two backcross generations with these genes for the following winter wheat cultivars: OK16D101089, Bob Dole, OK13625, KS090387K-20, Fortify, Tekoa and IDO676. Homozygous mutants for the TaGW2 gene in KSU cultivar KS080093K-18 were obtained for field trials in fall of 2023. The BC2F2 seeds were obtained for TaCKX6-1 in cultivar KS080093K-18 and BC2F1 seeds were received for TaCKX6-2­ in cultivar KS12DH0013-41. Transgene-free BSMV-based editing of wild relatives: Transfer of genetic diversity form wild relatives could be affected by non-adaptive variants of genes controlling domestication traits. One strategy is to edit domestication genes. To develop new tools for effective editing of wild relative genomes, the Cas9 gene construct was introduced into two wild emmer accessions, PI 466950 and PI 467008, by two rounds of backcrossing and marker selection. Plants were inoculated with BSMV constructs targeting the miR172 binding site in the Q gene (suite of domestication traits) and coding sequences of TaGW7 (grain size) and Btr1 (brittle rachis). In M1 plants, we have identified heterozygous gene editing events in TaGW7 (grain size) and Btr1 (brittle rachis). They are being increased to identify homozygous genotypes for field phenotyping. Additional rounds of editing are conducted to recover mutations in the Q gene. Editing regulatory regions of genes affecting grain yield and quality NAM-B1 gene explains 66% of the phenotypic variations in grain protein content (GPC). However, GPC increase in cultivars with functional NAM-B1 is often accompanied by low grain yield (GY). We aimed to generate new variation in the cis-regulatory region of NAM-B1 to adjust its expression and reduce trade-off between GY and GPC. Several approaches are being explored for promoter editing in wheat. 1) A total of 9 gRNAs were designed to 1.5 kb promoter region and sub-cloned into the Csy4-Cas9-GRF-GIF vector, 3 gRNAs for each construct. The pool of three constructs was used for biolistic transformation of line PI 664549 carrying NAM-B1. 2) We are using the BSMV-based method of editing to target the promoter region. For this purpose, we have transferred Cas9 locus into PI 664549 and inoculated plants with the BSMV constructs carrying 9 gRNAs as either single- or multiplexed (3 gRNAs per construct) expression cassettes. We have obtained several transgenic PI 466950 lines expressing multiplexed gRNAs. In T1 progeny of plant #3936, we have detected multiple deletions in the NAM-B1 promoter ranging from 200 to 500 bp. We have identified 8 lines with promoter deletions that do not show presence of Cas9 construct. These lines are being increased for assessing the impact of new cis-regulatory variants on gene expression and the process of nutrient remobilization from leaves to grain. For BSMV-based editing, 3 gRNAs separated by the Csy4 spacer were subcloned into BSMV. The T0 plant 3985-1 with high Cas9 expression were inoculated the BSMV transcripts. Analysis of somatic editing showed the presence of multiple edting events in the NAM-B1 promoter. These results indicate that Csy4 spacer could provide effecitive mean for multiplexing of BSMV-based editing. In fall of 2023, we expect to obtain M2 generation plants for phenotyping and expression analysis. Increase in the expression of SPL14 affects plant architecture and increases yield. SPL14 expression is repressed by miR156 whose binding site is conserved across all three homoeologs of SPL14 in wheat. Our goal is to edit the miR156 binding site and increase SPL14expression through creating in-frame shift mutations using BSMV-based genome editing approach. Two gRNAs were designed to target the miR156 binding site were subcloned into the BSMV construct. Out of all plants inoculated with BSMV, 11 showed somatic editing efficiency >10%. The M1 plants with fixed mutations will be increased for phenotyping. Editing of gliadin genes to reduce immunogenic response of wheat lines and improve bread-making quality: Gluten sensitivity caused by peptides produced during digestion of wheat products could be reduced by removing genes encoding gluten proteins. However, attempts to reduce gluten protein content by RNAi silencing and gene editing often led to reduction of end-use quality characteristics of wheat flour. In Year 1 of the project, we have developed a gene edited line #387-3-6 (Yu et al., 2023). The whole genome sequencing of #387-3-6 at 10x coverage demonstrated that all omega-gliadin gene copies and more than half of gamma-gliadin genes are edited or deleted. These results were consistent with the results of HPLC analysis of protein fractions, which showed reduction of these classes of gliadins. Using G12 and R5 antibodies we showed 47-fold reduction in immunogenic response of protein extracts prepared from line #387-3-6 compared to that from non-edited cultivar Fielder (Yu et al., 2023). Gliadins are part of gluten polymer network that defines the viscoelastic properties of dough. Using size-exclusion HPLC, we assessed the end-use quality characteristics of dough. Compared to non-edited lines, we observed significant increases in the ratio of glutenins to gliadins (from 1.29 to 1.53) and polymeric proteins to monomeric proteins (from 1.10 to 1.22), and in the percentage of glutenin macropolymer (from 51.6% to 53.2%). These results suggest that reduction of ω-gliadins and γ-gliadins facilitates gluten polymerization, which positively correlates with dough quality parameters. The dough quality tests were conducted using Mixograph and Farinograph confirmed this conclusion. Peak time in Mixograph substantially increased from 2.5 to 6.0 minutes, indicating that the amount of gluten macropolymer is higher in #387-3-6 than in non-edited line. For the results of Farinograph, there was a significant increase in dough strength. These results indicate that #387-3-6 carries novel gliadin alleles with reduced immunoreactivity and improved dough characteristics that could be used in breeding programs to reduce immunoreactivity and improve end-use quality traits in wheat.

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 omega- and gamma-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality, BioRxiv, 2023, doi: https://doi.org/10.1101/2023.01.30.526376
  • 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, 20(12):2332-2341.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. Leveraging modern genomic resources to identify adaptive variants and optimize breeding strategies in wheat. FROM SEED TO PASTA IV, Bologna - Italy, 26-29 October 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. CROP BREEDING INNOVATION HUB: NIFA IWYP Winter Wheat Breeding Innovation Hub (WWBI) at Kansas State University. 6th International Wheat Yield Partnership Program Conference, 4 - 5 October, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. Potential Application of High-Throughput Gene Editing in Wheat Breeding. 6th International Wheat Yield Partnership Program Conference, 4 & 5 October 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Akhunov E. CRISPR/Cas-based editing of wheat, U.S. Wheat Associates World Staff Conference, August 21 to 25, 2022 in Maui, HI, USA.


Progress 02/01/21 to 01/31/22

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:Due to COVID-19 related situation, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021. Due to low efficiency of the prime-editing technology and proved efficiency of the BSMV-based editing, we plan to switch to using the BSMV-based gene editing technology for producing mutations in the promoter of NAM-B1 and miRNA binding sites of the Q, TaSPL14 and TaSPL16 genes. What opportunities for training and professional development has the project provided?Two postdoctoral researchers (Wei Wang, Zitong Yu) attended the workshop "Barley and Wheat Transformation" organized by the Bill and Melinda Gates Foundation (June 15th, 2021). How have the results been disseminated to communities of interest?Stakeholders, farmers, breeders and industry representatives: PD Akhunov delivered two 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. 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. 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?Obtain BC3F1 generation plants with the introgression of genes encoding TaGW2, TaCKX6-1, TaCKX6-2, TaGW7 and TaARF4. Switch to BSMV-based gene editing technology for producing mutations in the promoter of NAM-B1 and miRNA binding sites of the Q, TaSPL14 and TaSPL16 genes. Produce M2 generation seeds with the fixed Cas9-induced mutations in these genes. Phenotypic evaluation of BSMV-edited lines for relevant agronomic traits, including grain size and number, grain weight, plant height, heading date, protein quantity and quality, etc. Educational activities: 1) 2-month training for undergraduate student Nick Stelling from ISU who joined PD Akhunov lab within the scope of the KSU REU project (https://www.plantpath.k-state.edu/undergraduate/reeu/2020-projects/index.html). 2) Deliver lectures on genomics and applications of genome editing to graduate students and postdoctoral researchers enrolled into the yearly Genomics Technologies workshop organized by the KSU Integrated Genomics Facility (https://www.k-state.edu/igenomics/workshops/Genomic%20Technologies.html).

Impacts
What was accomplished under these goals? Impact of COVID on the project: Due to COVID-19 related situation, lab closure and stay-in-home policy, production of genetic crosses and transgenic plants was significantly delayed in 2021. Introgression of edited alleles into adapted winter wheat germplasm: Introgression of the edited variants of genes affecting agronomic traits was performed using Bobwhite or Fielder cultivars with the edited TaGW2, TaCKX6-1, TaCKX6-2 and TaGW2, TaGW7 and TaARF4 genes. This spring, we have finalized second backcross (BC2F1) with these genes for the following winter wheat cultivars: OK16D101089, Bob Dole, OK13625, KS090387K-20, Fortify, Tekoa and IDO676. In addition, we have developed more advanced introgression lines (BC4F1) for TaGW2 in KSU cultivar KS080093K-18. The BC2F1 seeds were obtained for TaCKX6-1 in cultivar KS080093K-18 and BC1F1 seeds were received for TaCKX6-2­ in cultivar KS12DH0013-41. Editing genes associated with non-adaptive traits from wild relatives: Wild relative introgression is a powerful breeding tool, which with the development of genomic resources gained popularity. However, the efficiency of introgression is affected by non-adaptive variants of genes controlling domestication traits, such as threshability, spike compactness (both traits are affected by the Q gene) and brittle spikes (controlled by Btr1). We have introgressed the Cas9 locus into two wild emmer accessions, PI 466950 and PI 467008, with two rounds of backcrossing and marker selection. Plants carrying the Cas9 gene were inoculated with the BSMV constructs (Wang et al., 2022) targeting the miR172 binding site in the Q gene and coding sequence of Btr1. A total of 4 constructs were used per gene target. The effect of these edits on the domestication traits will be evaluated in the progeny of inoculated plants by the end of 2022. Novel transformation-free editing approach using the BSMV-based delivery of gRNA: We have established a transformation-free method for editing new targets in the wheat genome (Wang et al., 2022). The method relies on inoculation of Cas9-expressing wheat lines with Barley Streak Mosaic Virus RNA carrying gRNAs in the gamma chain of viral genome. We have demonstrated that multiplex editing could be performed using this method in the cultivars Fielder and Bobwhite, both transformed with the Cas9 gene. The method was first applied to generate heritable precise deletions in the promoter region of the AP2 transcription factor encoded by the Q gene and to perform multiplexed editing of agronomic genes (TaGW2, TaUPL3, TaGW7) controlling yield component traits. We also showed the Cas9 locus transferred into the winter and spring wheat cultivars by marker-assisted introgression supports effective editing in the adapted germplasm. We used this strategy to edit the TaGW7 gene in wheat line 3613474 from CIMMYT and Kansas winter wheat breeding line KS080093K-18 (Wang et al., 2022). The efficiency of somatic editing at TaGW7 in the leaf tissue after inoculation achieved 64%, with up to 17% of progeny carrying fixed mutations in all three homoeologs of TaGW7. This strategy is now being applied for generating edits directly in the adapted germplasm selected for this project. Since BSMV-sgRNAs can be designed to target hundreds of targets and inoculation procedure is simple, lines with the Cas9 introgression could be used for high-throughput functional screening in genetic studies aimed at identifying causal genes or novel variants affecting agronomic traits. Editing of gliadin genes to reduce immunogenic response of wheat lines and improve bread-making quality: Gluten sensitivity caused by peptides produced during digestion of wheat products could be reduced by removing genes encoding gluten proteins. However, previous efforts to remove gluten proteins by RNAi silencing and gene editing often led to reduction of end-use quality characteristics of wheat flour. In our study, we targeted only gamma- and omega-gliadins that are rich in toxic epitopes capable of causing celiac disease and wheat-dependent exercise-induced anaphylaxis (WDEIA). These genes encode smaller fraction of gluten proteins, and their removal should have reduced effect on end-use quality. The genes encoding gamma- and omega-gliadins are clustered at three homoeologous loci Gli-A1, Gli-B1 and Gli-D1 at the distal end of chromosome 1A, 1B and 1D short arm. A total of seven gRNAs targeting the repetitive regions of gliadin encoding genes on three chromosomes were designed and pooled together for the transformation of cultivar Fielder. Regenerated plants were screened by next-generation sequencing for the presence of all 7 gRNAs and Cas9 transgenes. So far, we screened 32 tillers of T0 plants using three-pairs of PCR primers, and identified one plant (#387-3-6) that carried mutations in gliadin-encoding genes from all three loci, Gli-A1, Gli-B1 and Gli-D1. The NGS of PCR amplicons confirmed mutations in five functional copies of omega-gliadins in Gli-D1, in one out of two functional copies of gliadin genes in each Gli-A1 and Gli-B1. Due to highly repetitive nature of the gamma-gliadin encoding genes, we experienced difficulty with assessing the number of edited gene copies by PCR and NGS. To characterize the full spectrum of the CRISPR-Cas9-induced mutations in line #387-3-6, we performed whole-genome sequencing of PCR-free NGS library to generate 10x genome coverage data. The NGS data is currently being aligned to the genome assembly of cultivar Fielder. Reverse-phase high performance liquid chromatography (RP-HPLC) was used to assess the amount of ω1,2-, ω5- and γ-gliadins in line #387-3-6. Compared to wild-type plant, in #387-3-6, the amount of ω-gliadins was decreased by 61.9%, with ω1,2-gliadins and ω5-gliadins decreased by 72.3% and 29.5%, respectively. The amount of γ-gliadin was decreased by 51.7%. Gliadins participate in the formation of gluten network through non-covalent binding that confers dough extensibility. The end-use quality characteristics of dough could be assessed by measuring the ratio of glutenins to gliadins (glu/gli ratio), the ratio of polymeric proteins to monomeric proteins (ratio of poly/mono) and the percentage of glutenin macropolymer (GMP%) using size-exclusion HPLC (SE-HPLC). Compared to wild-type, the gene edited Fielder cultivar showed statistically significant increase in the ratio of glu/gli from 1.29 to 1.53 and in poly/mono ratio from 1.10 to 1.22. The GMP% was slightly increased but not statistically significant, from 51.6% to 53.2%. These results suggest that the reduction of ω-gliadins and γ-gliadins facilitates gluten polymerization, which positively correlates with dough quality parameters. In addition, we used G12 and R5 antibodies raised against immunoreactive peptides in omega- and gamma-gliadins to perform ELISA and assess the immunogenic response of protein extracts prepared from line #387-3-6. Preliminary results showed that both antibodies detect more than 80% reduction in reactivity compared to non-edited cultivar Fielder. Overall, our results indicate that #387-3-6 carries novel gliadin alleles with reduced immunoreactivity and improved dough characteristics that could be used in breeding programs for improving end-use quality traits in wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang W, Yu Z, He F, Bai G, Trick H, Akhunova A, Akhunov E. Multiplexed promoter and gene editing in wheat using the virus-based guide RNA delivery system. BioRxiv, 2022, DOI: https://doi.org/10.1101/2022.04.06.484365.
  • Type: Other 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: Other 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: Other 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: Other 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.