GENOME EDITING FOR IMPROVING WHEAT YIELD AND YIELD-RELATED TRAITS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1011365
• Grant No.: 2017-67007-25932
• Project No.: KS1011667
• Proposal No.: 2016-06711
• Program Code: A1142
• Project Start Date: Nov 15, 2016
• Project End Date: Nov 14, 2020
• Grant Year: 2019

Project Director
Akhunov, E. D.

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

Performing Department
Plant Pathology

Non Technical Summary
The rate of yield gain in breeding programs lags behind the demand for crop production forcing the community of breeders and geneticists to investigate innovative strategies to break this negative trend. Genetic mapping studies in crops, fueled by the availability of whole genome sequences, identified a number of genes controlling major agronomic traits including yield components. The release of the annotated genome sequence and the development of functional genomics tools for wheat now provides the opportunity to easily extrapolate gene mapping information from other crops to wheat.CRISPR/CAS9-based genome editing has emerged as a disruptive technology that can take full advantage of the available genomic information and help to rapidly validate gene candidates identified by the inter-species comparative analyses, or help to transfer traits among wheat cultivars. The technology relies on the ability of short sequence called guiding RNA (gRNA) to guide enzyme CAS9 capable of cutting DNA to a precise location in the genome. The CAS9/gRNA complex then acts as a pair of scissors to cut DNA and introduce changes into the genetic code of specific genes. Our project will explore the capabilities of this novel CAS9-based gene editing technology to unlock the potential of the complex wheat genome, thereby building a foundation for transformative approaches to wheat improvement that can be deployed in both the public and private sectors.We have identified 18 genes that in rice or maize that have a potential to affect yield component traits in wheat. We will use CAS9-based editing technology to modify these genes in wheat cultivars and then assess whether the introduced changes in genes result in positive changes in the yield component traits. This will allow us not only to create wheat lines with the improved yield components but also to optimize the CAS9-based technology for manipulating the wheat genome. This knowledge will be critical for devising future gene editing strategies to improve other agronomic traits in wheat. For the newly developed variants of genes affecting yield components we will initiate the transfer into the adapted cultivars grown in Kansas and Oklahoma. These efforts will help us to assess the value of gene editing for wheat improvement and establish a pipeline for deploying this technology in wheat breeding programs.The project will train a new generation of breeders and geneticists in genome editing technology and the regulatory aspects of crop biotechnology thereby contributing to fulfilling the needs of industry, stakeholders, and academia in a trained workforce and making wheat production more profitable. By pursuing these objectives, the project will address one of the key aims of the NIFA-IWYP to increase the genetic component of wheat yield and achieve sustainable intensification of agricultural production. The project is closely integrated with the Wheat CAP and is a part of overarching International Wheat Yield Partnership program aimed at increasing the genetic yield potential of wheat using innovative approaches.

Animal Health Component

0%

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	100%

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

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1080 - Genetics;

Keywords

crispr/cas9

biotechnology

genome editing

wheat yield

Goals / Objectives
The analyses of yield trends in major crops including wheat over the period of 1961-2008 has revealed the lack of significant progress in 24-39% of global crop growing areas. This negative trend combined with growing demand for wheat, which provides 20% of the daily protein and of the food calories for 4.5 billion people (FAO), necessitates the adoption of innovative approaches for developing high-yielding varieties. Recent progress in understanding the genetics of yield components in different crops provides the foundation for developing new strategies for increasing yield potential in wheat. This trait has a complex genetic basis that is controlled by a number of genes that contribute to the different trait components including the number of spikes per plant, number of spikelets per spike, number of grains per spikelet, and grain size and weight. Recent studies showing that the homologous genes of wheat and rice can affect yield components in both crops suggest that the targeted manipulation of these genes using the genome editing technologies hold a great promise to improve wheat yield.Genome editing has emerged as a powerful tool for creating novel variation in the targeted genomic regions. This technology relies on the ability of the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)-associated protein (CAS) CRISPR/CAS9 system to introduce double-stranded breaks (DSB) into DNA that are then repaired through either 1) error-prone non-homologous end joining (NHEJ) mechanism resulting in deletions and single base substitutions in DNA sequence, or 2) homology-directed repair (HDR). The HDR can be used for precise sequence insertion or replacement through recombination with the exogenously supplied "donor DNA". The CAS9 nuclease can be directed by a single-guide RNA (sgRNA or gRNA) that contains a 20 nt guide sequence to any complimentary genomic regions and generate mutations, delete, add, or replace segments of the genome.The recently released wheat genome sequence now makes it possible to identify candidate genes for editing by the comparative analyses of wheat with other crops. The identified gene candidates can be validated using the wheat TILLING populations, or gene mapping resources developed by our team as a part of the TCAP. Our group has established and tested the CRISPR/CAS9-based system for targeting single and multiple genes in polyploid wheat. These resources provide a unique opportunity to develop a CAS9-based platform for studying the gene function in wheat and introducing edited genes into the wheat breeding pipeline. In this project we outline a plan for developing public genomic resources for editing genes controlling yield components in wheat and evaluating different strategies for editing targets in the polyploid genome. To facilitate technology adoption by breeding programs, we established collaboration with the Wheat CAP on studying the genes controlling yield QTL and training a new generation of breeders and geneticists in genome editing technology. Our project team is a part of the IWYP and will share resources, genetic material and expertise with other IWYP partners. We also collaborate with the UK IWYP project team to characterize the candidate genes selected for gene editing in our project using their TILLING populations.Our grant proposal will be aimed at developing effective strategies and resources for the CAS9-based editing of the wheat genome with the goal of targeting or regulating genes affecting yield components. The long term objective of the proposal is to establish a pipeline for deploying genome editing in wheat breeding to increase yield potential and to train the next generation of geneticists in the application of genome editing for improving wheat. Objective 1: Identify gene targets for editing and select the optimal genome editing strategy depending on the biological function of a gene.A number of genes controlling phenotypes affecting yield have been identified in rice, maize and wheat. The homologs of some of these genes were shown to have alleles with a positive effect on wheat yield components suggesting that many traits in cereals have similar genetic basis, and that by targeting these genes we have a high chance to improve wheat yield potential. These genes will be selected for targeted gene editing in wheat and assessing the effect of editing on yield components. Depending on the biological function of the selected candidate genes different editing strategies will be developed and tested.Objective 2: Develop and optimize the CRISPR/CAS9 constructs for different genome editing scenarios in the polyploid wheat genome. While it is relatively easy to reprogram CAS9 to target different sites in a genome by changing the sgRNA sequence, the efficiency of editing, on- and off-target activities can vary from sequence to sequence. We will combine bioinformatics-assisted design using the existing tools with the high-throughput screening of multiple sgRNAs using the expression in wheat protoplasts and wheat embryo for assessing the ability of gRNAs to guide CAS9 to a correct site.Objective 3: Develop and validate the CRISPR/CAS9-expressing transgenic spring and winter wheat lines. In this objective, two spring and two winter wheat cultivars will be transformed using the gRNA-CAS9 constructs and transgenic plants will be the presence of editing events by the next-generation sequencing of targeted genomic regions.Objective 4: Phenotype transgenic lines and the TILLING mutants for yield components and transfer the CAS9-edited gene variants to the genetic backgrounds of locally adapted wheat cultivars to initiate the deployment of novel variation in wheat breeding programs. Transgenic lines carrying the edited version of genes and the TILLING mutants of cultivars Kronos and Cadenza will be grown in both greenhouse and field conditions, and phenotyped for a set of yield component traits including number of fertile tillers/spikes per plant or per unit area, average number of kernels per spike, and average kernel weight. Within the timeframe of the project we will initiate the transfer of the best performing edited gene variants to adapted cultivars.Objective 5: Train the next generation of breeders in genome editing strategies and communication skills to advocate for the utility of genome editing technology in breeding and improving food security.To accelerate the acceptance of genome editing tools for crop breeding, our project in collaboration with the Wheat CAP will develop educational modules for postdoctoral researchers and PhD students covering the basics of the CAS9 editing technology and strategies. An important aspect of preparing the next generation of plant breeders is the ability to communicate the sensitive issues of biotechnology and genome engineering to policy makers and the broader public. To address this aspect of training, we have partnered with The Cornell Alliance for Science (allianceforscience.cornell.edu), an initiative focused on building an international network of concerned scientists, farmers, and humanitarian organizations working to restore the place of science in food and agricultural policy decisions.

Project Methods
Recent studies showing that the homologous genes of wheat and rice can affect yield components in both crops suggest that the targeted manipulation of these genes using the genome editing technologies hold a great promise to improve wheat yield. We have identified the wheat orthologs of yield component genes mapped in rice and maize by comparing with the reference genome. The beneficial (positive effect on yield components) alleles for 8 of these genes were loss-of-function mutations or variants with reduced gene expression, 8 more genes were variants with increased gene expression, and the last 2 genes were nonsynonymous mutations affecting enzymatic activity of encoded proteins. The beneficial (positive effect on yield components) alleles of selected genes were loss-of-function mutations or variants with reduced gene expression, variants with increased gene expression, or nonsynonymous mutations affecting enzymatic activity of encoded proteins. Out of 18 gene targets selected in our project for editing five genes (TaCKX6, TaGS-D1, TaTGW6, TaGW2, TaCWI) have been shown to affect thousand grain weight (TGW) in multiple environments and the components of yield in both wheat and rice (Zhang et al. 2012, 2014; Jiang et al. 2015; Ma et al. 2015; Hanif et al. 2016; Simmonds et al. 2016). The effect size of the allelic variants of these genes in wheat was substantial. For example, the GW2-A1 mutant allele significantly increased TGW (6.6 %), grain width (2.8 %) and grain length (2.1 %) in tetraploid and hexaploid wheat across 13 experiments.Thus, out of 18 selected genes, the editing of these five will have effect on yield and will allow us to test major gene editing strategies outlined in the project. Considering the fact that nearly 30% of cloned genes affecting yield components in rice were also shown to affect yield in wheat, we believe that significant fraction of the remaining genes in our list will have effect not only on yield components but also on yield. Additional yield component genes for editing will be selected among the candidates identified in the QTL mapping projects to be performed by the Wheat CAP team.In addition, our project will collaborate with the UK IWYP-funded project led by C. Uauy (John Innes, UK). The phenotypic effects of knock-out mutations in the candidate genes selected for gene editing in our project will be studied using the wheat TILLING populations. Once available, this information will be used in our project to prioritize gene targets for editing or gene-edited wheat lines for further phenotypic characterization.Different genome editing strategies (gene knockout, activation of gene expression, suppression of gene expression or sequence replacement) will be applied depending on the nature of a positive alleles affecting the yield component. All strategies have been previously tested in wheat, maize or rice and we expect no major problems with accomplishing the project objectives.We will combine bioinformatics-assisted design using the existing tools with the high-throughput screening of multiple sgRNAs using the expression in wheat protoplasts and wheat embryo for assessing the ability of gRNAs to guide CAS9 to a correct site. Our previous analyses showed that the E-CRISP and sgRNA tools provided the good predictions of gRNA editing capability consistent with their ability to modify DNA in wheat protoplasts. We will apply these tools for selecting gRNAs targets in the wheat genome. In addition, newly developed web-based tools integrating multiple gRNA scoring systems (http://crispor.tefor.net/) will be tested for designing gRNAs for wheat (Haeussler et al. 2016). The CRISPOR web-based tool accepts new genomes for inclusion into the gRNA design database. The project team will collaborate with the CRISPOR tool developers on integrating the latest version of the wheat genome assembly into the list of available genomes. The experimental validation of gRNA constructs will be performed by expressing all gRNA constructs in the wheat protoplasts and transformed wheat embryos followed the next-generation sequencing of amplicons obtained for the edited genomic regions. Both approaches were tested by the project team. The gRNA/CAS9 constructs will be designed to test different genome editing strategies including sequence replacement, mutagenesis, and CAS9-guided gene expression activation and suppression.The gRNA/CAS9 constructs will be transformed into spring wheat cultivars 'Fielder' and 'Bobwhite', and winter wheat cultivars 'Jagger' and '2174'. All constructs will contain or be co-bombarded with the bar gene for transformant selection on the selective media with glufosinate. Each herbicide resistant tiller of a transgenic event will be tested for the presence of CAS9 by PCR; CAS9-positive tillers will be tested for the expression of CAS9 and gRNA by qRT-PCR. The transgenic plants with duplicated genes, modified regulatory sequences, or expressing dCAS9 fusions will be tested for the level of edited gene expression compared to the expression level of a gene under the control of the original promoter. Mutations in the targeted regions will be identified by the next-generation sequencing (NGS). The sequence addition or replacement events will be validated by PCR with the primers designed to amplify the boundaries of insertions sites, followed by the NGS of amplicons.Transgenic lines carrying the edited version of genes and the TILLING mutants of cultivars Kronos and Cadenza with mutations in the candidate genes will be grown in both greenhouse and field conditions. Field phenotyping will be performed on wheat lines grown in two locations in Kansas and Oklahoma. The phenotyping data will be collected for three major components that affect the grain yield in wheat: number of fertile tillers/spikes per plant or per unit area, average number of kernels per spike, and average kernel weight. The kernels per spike will be dissected into two subcomponents: spikelets per spike and kernels per spikelet. Within the timeframe of the project we will initiate the transfer of the best performing edited gene variants to adapted cultivars. Transgenic wheat lines expressing the CAS9/gRNA construct in the background of Jagger or Bobwhite cultivars and carrying edited gene variants will be crossed with adapted cultivars from Kansas and Oklahoma. In the crosses, lines that have the edited gene variants but lack the CAS9/gRNA construct will be selected and used to generate the DH lines at the HPI center (Kansas, Manhattan).To accelerate the acceptance of genome editing tools for crop breeding, our project will develop educational modules for postdoctoral researchers and PhD students covering the basics of the CAS9 editing technology and strategies. Precision Breeding Advocacy and Communications workshop organized by the Cornell Alliance for Science will introduce students and postdoctoral researchers employing this technology to skills in advocacy and communications. The workshop will prepare project scientists to be effective science advocates that will have impact beyond the scope of this project. The educational and training activities in the project will be coordinated with the workshops and project meetings organized by the Wheat CAP to minimize travel expenses and maximize inter-project coordination. The project participants will develop training modules for online courses delivered through the Plant Breeding Training Network in coordination with the Wheat CAP. The courses will cover various technical aspects of genome editing and the utility of this technology for improving crops. Through these courses students participating in the PBTN's education courses will have the opportunity to interact with the experts in genome editing, crop breeding, and scientific communication.

Progress 11/15/16 to 11/14/20

Outputs
Target Audience: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. In addition, the project's objectives are aligned with the priorities of the Wheat CAP that includes an Industry Liaison Group, representatives in the National Wheat Improvement Committee, and breeders which coordinates research priorities with NAWG and Wheat Associates. PD Akhunov presented wheat genome editing technology for the invited staffers supporting Kansas' congressional offices and selected citizen volunteer advocates. (September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA). Also, PD Akhunov discussed the potential of gene editing technology for crop improvement at the Ag Innovation Forum organized by the Agricultural Business Council of Kansas City (June 14, 2017, Kansas City, KS). Project results are also presented to wheat researchers, breeders and industry representatives at the International Wheat Yield Initiative meeting in CIMMYT, Obregon, Mexico. General public: We collaborated with The American Seed Trade Association (ASTA) and KSU communication department on creating two videos that featured the project team's members discussing the potential of gene editing technology for wheat improvement. The video material was broadly distributed to general public through YouTube and Vimeo platforms. In addition, the main findings of the project were highlighted in the press releases prepared by the KSRE communication department. Wheat researchers, breeders, industry representatives: Project results are also presented to wheat researchers, breeders and industry representatives at the International Wheat Yield Initiative meetings (Obregon, Mexico and John Innes Center, UK). The project team presented their discoveries to wheat researchers and breeders at the national and international conferences: 1) International Wheat Conference in Saskatoon (Canada), 2) Plant and Animal Genome (San Diego, CA), 3) National Association of Plant Breeders Meeting (Pine Mountain, GA), and 4) In Vitro Biology Meeting (Tampa, FL). The results of the project were disseminated to the international wheat community by publishing in the international peer-reviewed journals. Students and postdoctoral researchers: An important aspect of preparing the next generation of plant breeders is the ability to communicate the sensitive issues of biotechnology and genome engineering to policy makers and the broader public. To accelerate the acceptance of genome editing tools for crop breeding, our project in collaboration with the Cornell Alliance for Science organized workshop (Speaking Science: CRISPR, Consumers and Conversations, Jan. 8-9, 2019, San-Diego CA) for postdoctoral researchers and PhD students covering the basics of the CRISPR editing technology and strategies, and prepare graduate students and scientists to be effective science advocates that will have impact beyond the scope of this project. Our project developed educational modules for postdoctoral researchers and PhD students covering the basics of the CAS9 editing technology and strategies, and prepare graduate students and scientists to be effective science advocates that will have impact beyond the scope of this project. In coordination with the Wheat CAP, we organized a workshop at UC Davis, Kansas State University and Cornell University. During these workshops, co-PD S. Evanega covered various aspects of communicating the gene editing technology to public. PD Akhunov delivered a lecture and provided training in using the gene editing technology for functional validation of genes and generating beneficial allelic diversity for improving wheat. The KSU team also participated in the two month REEU program run the KSU Department of Plant Pathology by hosting an undergraduate student. Changes/Problems:Due to COVID-19 related situation, phenotypic evaluation of gene edited plants was not accomplished in 2020, as was planned originally. The project team will finalize phenotypic analyses of these plants within the scope of the Wheat CAP (E. Akhunov is co-PD of Wheat CAP) and other gene editing projects funded by USDA NIFA. In 2020, E. Akhunov received funding from USDA NIFA to continue gene editing projects in wheat. What opportunities for training and professional development has the project provided?In collaboration with the Cornell Alliance for Science, we have organized a workshop "Speaking Science: CRISPR, Consumers and Conversations", Jan. 8-9, 2019, San-Diego, CA. The course was organized along-side the Plant Animal Genome meeting and attended by nearly 30 graduate students from the US wheat breeding programs involved in Wheat CAP. Project team shared their a Gene Editing module for the Genomic Technologies Workshop held at KSU June 17-21, 2019 by the KSU Integrated Genomics Facility. The workshop was attended by 18 graduate students. In January of 2018, in coordination with the 2018 Wheat CAP workshop at the University of California Davis, our team organized a Gene Editing and science communication workshop. S. Evanega provided a two-hour primer on science communication, and why community engagement and science communication around editing is essential for its success. Akhunov provided a lecture on the basics of gene editing in wheat and hands-on training in bioinformatical tools used for designing gene editing experiments. This set the stage for a larger science communication workshop called "Speaking Science" that was held at Cornell University June 25-29th 2018. Our project supported ten WheatCAP graduate students from eight US land grant institutions to attend the course. The course featured a series of lectures on science communications topics as well as several practical components, including engaging with tough journalists, sticking to message, and developing and delivering your personal science story to connect on shared values. Each participant ended the course having created an output in one of five tracks: how to write an op-ed, radio interview, video interview, print media interview, or social media strategy. Several of these pieces were aired or printed or are still under development. A number of students in the Op-ed track wrote opinion pieces on the USDA proposed bioengineered label, as that public comment period was open and on the cusp of closure. Example of participant output (print media track): https://www.kansasfarmer.com/crops/k-state-grad-student-finds-herpassion- study-wheat-genetics?NL=FP-008&Issue=FP-008_20180712_FP-008_220&sfvc4enews=42&cl=article_1_b&utm_rid=CPG02000000752256&utm_campaign=29336&utm_medium=email&elq2 =665b75b943604237aae1f9eee0456529 Participants have also written about their experience in the workshop. One of the pieces was publlished on the AAAS website: https://www.aaas.org/blog/public-engagement-reflections/sharing-science-through-storytelling Following the workshop, participants offered feedback on the course in a focus group as well as through a post course survey. Students ranked exceptionally high the story telling element of the workshop as well as the mock interview with journalists. On a scale from 1 to 5 with 5 being most confident, participants on average cited an increase from 2.8 to 4.3 in their level of confidence when communicating to non-specialist audiences about their work. Postdoctoral researchers W. Wang and B. Tian made oral presentations about their projects at the national and international meetings, and also at a departmental seminar series. B. Tian supervised for 2 months an undergraduate student from the KSU REEU program. Q. Pan from KSU defended her MS thesis on application of gene editing technology for wheat improvement. Currently, Q. Pan works in the biotech industry; B. Tian obtained a new position and now oversees a plant transformation facility in a biotech company. An undergraduate student from KSU, A. Dunnivan, now joined Akhunov Lab as a laboratory manager. The team from Oklahoma State University trained two postdoctoral researchers, Dr. Ragupathi Nagarajan, and Dr. Lei Lei. Three Ph.DD students, Forrest C.C. Kan, Xiaoyu Zhang, and Lei Lei were trained and obtained skills and expertise in generation of gene editing plants using the CRISPR/CAS9 platform established in this project. Lei has graduated in 2018 and Forrest and Xiaoyu graduated in 2019. How have the results been disseminated to communities of interest?The results generated by the project are presented to a broader public and private wheat research and breeding community through the publications in the peer-reviewed journals and presentations at the international and national meetings and on-line seminars. PDs prepared several press releases highlighting the discoveries made in the project. PD Akhunov collaborated with the KSU communication department to prepare video material about the project that was deposited to KSRE YouTube video repository. The current state of wheat genome editing initiatives at KSU have been presented to industry representatives and stakeholders through a series of presentations at the Kansas Wheat Innovation Center. PD Akhunov collaborated Kansas Wheat Commission and ASTA in creating movie titled "Plant Breeding and Innovation Part 3: Breeding for Better Bread" that was broadly distributed through the YouTube Channel. PD Akhunov presented wheat genome editing technology for the invited staffers supporting Kansas' congressional offices and selected citizen volunteer advocates (September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA). Also, PD Akhunov presented and discussed the potential of gene editing technology for crop improvement at the Ag Innovation Forum organized by the Agricultural Business Council of Kansas City (June 14, 2017, Kansas City, KS). The project results and material on gene editing are incorporated into the course material (Introduction to Systems Biology) developed at the Kansas State University. The gene editing plasmid constructs developed by the project are distributed to research collaborators. The gene edited wheat lines have been shared with collaborators for evaluation in the field conditions. Project directors in colboratoion with Cornell Alliance for Science and Wheat CAP organized three workshops at UC Davis, KSU and Cornell to train graduate students in gene editing technology and science communication skills. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Targeted yield component genes: We have created wheat carrying the CRISPR-induced variants in 11 genes that have potential to affect yield component traits: TaGW2, TaCKX2-1, TaCKX2-2, TaGS3, TaTGW6-1, TaARF4, TaGW7, TaAn-1, TaLAC1-1, TaLAC1-2 and TaGASR7. TaGW2: The TaGW2 gene homoeologues is a negative regulators of grain size and thousand grain weight (TGW) in wheat. Using both CRISPR and TILLING mutagenesis, we developed single-, double- and triple-gene knockouts of TaGW2. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases of 16.3% (gene edited) and 20.7% (TILLING) in TGW (Wang et al., 2018a, Wang et al., 2018b). TaGW7: By CRISPR editing of the TaGW7 gene, a homolog of rice GW7 encoding TONNEAU1- recruiting motif protein, we demonstrated that this gene controls grain weight and dimension traits in wheat (Wang et al., 2019). Triple mutants increased grain weight by 8.9%, grain width by 5.8% and reduced grain length by 2.2%. The plant height (14.4%) and flag leaf length (12%) are reduced dramatically while flag leaf width (11.8%) is increased in the triple mutants. TaGS3: Using CRISPR-Cas9 technology, we modified the TaGS3 homeologs in all three genomes, and showed that these mutations increase grain weight by 3-4%, and have relatively small effect of grain dimension traits. The manuscript presenting the evaluation of the TaGS3 mutants is prepared for submission. TaCKX2-1 and TaCKX2-2: The triple knock-out of TaCKX2-1 was crossed with the high-biomass CIMMYT line 3613474. Phenotyping of 132 F2 plants showed that plants carrying 3-5 mutated alleles of gene exhibit 10% (P< 0.01) increase in grain number per spike compared to plants carrying 0-2 mutated alleles. The TaCKX6-2 mutants were crossed with two winter wheat cultivars KS12DH0013-41 and KS080093K-18. The phenotypic effects were evaluated in the F2 population derived from KS080093K-18. The knock-out of the A and B genome homoeologs increases number of grains per spike by 3.6% and TGW by 2.4%. TaARF4: This gene encodes for an auxin-responsive factor controlling many aspects of plant growth and development. In wheat, there are six alleles (12 copies) of the ARF4 gene on chromosome 3. Currently, we evaluated a T2 generationCRISPR-edited wheat lines with mutations in the B genome of only one of the genes, which resulted in 3% increase in TGW. A T0 plant 7388-1 carrying mutations in all 12 copies of ARF4 was crossed to Bobwhite and will be evaluated by fall of 2021. TaTGW6-1: The TGW6-1 gene mutant line 5023-2 was crossed with cultivar Bobwhite and F3 population derived from this cross will be phenotyped by fall of 2021. TaLAC1: This gene has paralogs on chromosomes 1 and 3. Phenotyping performed using 100 T2 TaLAC1 mutants showed no phenotypic effects on grain size and number. The mutants of TaLAC1 on chromosome 3 were obtained. Phenotypic analyses will be performed in 2021. TaAn1: The preliminary analyses of the CRISPR-edited lines with TaAn1 gene mutations showed ~10% decrease in grain weight. We have also created transgenic plants overexpressing the TaAn1 gene under the control of ubiquitin promoter. This transgene will be used for investigating the effect of the TaAn1 product increase on yield component traits. Transgenerational activity of CRISPR-Cas9 facilitates multiplexed gene editing in wheat: During multiplex gene editing not all copies of genes in the polyploid genome can be edited due to low frequency of editing events in transgenic plants. We showed that the targets not subjected to editing at earlier generations of transgenic plants could be edited in the next generations. The study reporting this finding was published in the inaugural issue of The CRISPR Journal (Wang et al., 2018). Increasing the efficiency of gene editing in wheat: One of the main limitations of CRISPR-CAS9 gene editing in wheat is its low efficiency. Our team has identified transgenic Bobwhite line 707-1 expressing the CRISPR construct at a high level, four times higher than the level of the Actin gene expression. This line was tested for the ability to induce novel gene editing events in target genes of the F1 hybrids. We showed that the level of Cas9 expression is one of the factors defining the gene editing frequency. Currently, we are using lines high-expressing Cas9 lines for inducing mutations in the F1 hybrids by crossing with the recipient germplasm. Complexes of yield component genes: To investigate the effects of gene editing in several genes on yield component traits, in the progeny of crosses among lines carrying single-gene mutations in TaGW2, TaGASR7, TaAn-1, TaCKX2-1, and TaCKX2-2 we selected plants with different combinations of edited gene pairs. Preliminary phenotyping data for F2 population carrying mutations in the TaGW2-TaCKX6-1 genes show ~10% increase in the grain number per spike. Phenotyping of the F3:4 generation double-gene mutant plants for yield component traits was planned for the fall of 2020, but will be accomplished only in summer of 2021 due to COVID19-related delays. To increase the multiplexing capacity of gene editing constructs, we have established the Cpf1-based system for wheat. Cpf1 has different PAM and requires shorter 40-44 bp-long CRISPR RNA. In the wheat protoplasts, our Cpf1-based multiplex construct showed editing efficiencies comparable to that of Cas9. We have used a Cpf1-based construct targeting a complex of gene TaGASR7-TaGW7-TaAn-1-TaSPL16-TaGW2-TaGS3-TaGSE5 to transform cv. Bobwhite. The phenotypic effects of one of the genes from this array (TaGW7) was confirmed (Wang et al., 2021). Transfer of gene editing events to target cultivars: Previously, we have demonstrated that wheat lines expressing the CRISPR-Cas9 construct at high levels has the ability to induce novel gene editing events in the homologous gene copies of the F1 hybrids. We used this strategy to introduce mutations into the TaGW2, TaGS3, TaCKX6-1, TaCKX6-2, TaARF4, TaTGW6, and TaGASR7 genes of five cultivars from the CIMMYT and Kansas wheat breeding programs. The direction of the effects of TaGW2 and CKX2-1 mutagenesis on phenotype in the background of CIMMYT or Kansas wheat cultivars were consistent with those observed in original lines, suggesting the utility of this strategy for improving wheat. We also generated CRISPR mutations in winter wheat (Gallagher, Smith's Gold, OK13209, Jagger, and 2174) by targeting TaGW2, TaGASR7, TaGSE5, TaBH1, TaVRN1, and TaE3. The phenotypic evaluation is underway. The testing of T1 progeny of the plants carrying the edited TaBH1 gene showed decrease in plant height and increase in the number of spikelets. Field phenotyping: We have performed field-based evaluation (Pullman, WA; Ashland, KS) of the spring and winter wheat lines with the edited copies of the TaGW2 gene. Plants has been tested to be free from the CRISPR construct. In the gene-edited wheat lines with all three copies of the gene mutated, we found significant increase in thousand grain weight (15%) and grain protein content (8- 10%), but no significant increase in grain yield. Workshops: In collaboration with the Cornell Alliance for Science and Wheat CAP, we have organized three workshops. The workshop were focused on how to talk to audiences about gene editing, incorporating the communications and messaging data collected by the Center for Food Integrity's Gene Editing Coalition. In these courses, participants reviewed the data on gene editing messages, talked about which analogies work, and which do not, and developed a consistent core message for broad audiences. In total, the courses were attended by nearly 30 graduate students from the US wheat breeding programs. Project participants also helped to develop a training module on Gene Editing for the Genomic Technologies Workshop held at KSU June 17-21, 2019 by the KSU Integrated Genomics Facility. The workshop was attended by 18 graduate students.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, W., Akhunova, A., Chao, S. & Akhunov, E. Optimizing multiplex CRISPR/CAS9Cas9 system for wheat genome editing. BioRxiv, 2016, https://doi.org/10.1101/051342
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang W, Simmonds J, Pan Q, Davidson D, He F, Battal A, Akhunova A, Trick HN, Uauy C, Akhunov E. Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat. Theor Appl Genet. 2018 Nov;131(11):2463-2475.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang W, Pan Q, He F, Akhunova A, Chao S, Trick H, Akhunov E. Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. CRISPR Journal, 2018, 1, 65-74.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang W, Pan Q, Tian B, He F, Chen Y, Bai G, Akhunova A, Trick HN, Akhunov E. Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat. 2019 Oct;100(2):251-264.
• 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. BioRxiv, 2021, https://doi.org/10.1101/2020.12.09.418624
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Q. Pan. Application of CRISPR/Cas9 to edit genes affecting seed morphology traits in wheat. M.S. Thesis, 2019, Kansas State University
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu M, Lei L, Miao F, Powers C, Zhang X, Deng J, Tadege M, Carver BF, Yan L. 2017. The dicot Medicago gene STF increased leaf width in monocot wheat. 13th International Wheat Genetics Symposium, April, 2017. Tullin, Austria.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, Pan Q, He F, Akhunova A, Evanega SD, Yan L, Trick H, Akhunov E. Genome editing for improving wheat yield and yield-related traits. 2nd IWYP Conference, 20-23 March, 2017, Obregon, Mexico.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu M, Lei L, Miao F, Powers C, Zhang X, Deng J, Tadege M, Carver BF, Yan L. 2017. The STENOFOLIA gene from Medicago alters leaf width, flowering time and chlorophyll content in transgenic wheat. Plant Biotechnol J. 2018 Jan;16(1):186-196.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, Pan Q, He F, Akhunova A, Evanega SD, Yan L, Trick H, Akhunov E. Genome editing for improving wheat yield and yield-related traits. National Association of Plant Breeders, August 7-8 2017, UC Davis, Davis, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: E. Akhunov. Application of Multiplex CRISPR/Cas9-Based Genome Editing Strategy for Targeting Multiple Agronomic Genes in Wheat. 2017, ASA, CSSA, and SSSA Annual Meeting in Tampa, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: E. Akhunov. Wheat genome editing. Presentation for the invited staffers supporting Kansas congressional offices and selected citizen volunteer advocates. September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Akhunov E. The Potential for Gene-editing in Cereal Crops, Ag Innovation Forum, The Agricultural Business Council of Kansas City. June 14, 2017, Kansas City, KS.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, He F, Pan Q, Chao S, Trick HN, Akhunova A, Akhunov E. Application of Multiplex CRISPR/Cas9-Based Genome Editing Strategy for Targeting Multiple Agronomic Genes in Wheat. Plant and Animal Genome meeting, Jan 14-18, 2017, San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wang W, Pan Q, He F, Fernandez de Soto M, Ren J, Chen Y, Tian B, Akhunova A, Chao S, Trick HN, Akhunov E, Application of CRISPR/Cpf1-Based Genome Editing in Polyploid Wheat, Jan 13-17, 2018, San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pan Q, He F, Akhunova A, Chao S, Trick HN, Akhunov E. Application of CRISPR/Cas9 technology to analyzing gene function in the wheat genome and improving agronomic traits, Nebraska Plant Breeding Symposium, March 13th, 2018, University of Nebraska, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Wheat Improvement through Gene Editing, Flowers Foods and Grain Craft, June 6, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Gene Editing Opportunities and Capacities at KSU, Kansas Wheat Meeting, May 8, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. CRISPR/Cas9 based wheat genome editing, Wheat CAP workshop, Jan 8th-12th, 2018 University of California, Davis, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Editing the wheat genome, Sept. 19-21, 2018, International Conference From Seed to Pasta III, Bologna, Italy
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: L. Yan, W. Wang, H. Trick, Akhunov E. Gene editing for improving wheat yield component traits, 3rd Annual IWYP Program Conference, June 2018, John Innes Center, UK.
• Type: Other Status: Published Year Published: 2018 Citation: Akhunov E. Update on Gene Editing initiatives in wheat at KSU, Kansas Wheat Innovation Center meeting with TechAccel, Aug. 22, 2018, Kansas Wheat Innovation Center, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: W. Wang, Q. Pan, Y. Chen, B. Tian, F. He, A. Akhunova, S. D. Evanega, L. Yan, H. Trick, E. Akhunov Applications of Gene Editing to Improve Yield Component Traits in Wheat. In Vitro Biology Meeting, June 8-12, 2019, Tampa, Florida
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Wheat Gene Editing Platform (WGEP) for improving Kansas Wheat, KWC Research Committee meeting, March 21, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wang W, Tian B, Pan Q, Chen Y, He F, Akhunova A, Evanega S, Yan L, Trick H, Akhunov E, NIFA: CRISPR-Based Precision Breeding in Wheat. National Association of Plant Breeders Meeting, August 25-29, 2019, Pine Mountain, GA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wang W, Tian B, Pan Q, Chen Y, He F, Akhunova A, Evanega S, Yan L, Trick H, Akhunov E, CRISPR-Based Precision Breeding in Wheat, 1st International Wheat Conference, July 21-26, 2019, Saskatoon, Canada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E. Akhunov, Wheat Gene Editing Platform (WGEP) for improving Kansas wheat, Kansas Wheat Commission, 2019, March 28, 2019, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: W. Wang, E. Akhunov, CRISPR-Cas9 genome editing technology as a tool for precision breeding in wheat. KSU Fall Seminar Series, 2019, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: He F, Wang W, Rutter WB, Akhunova A, Fernandez de Soto M, Szabo LJ, Jordan K, Rouse M, 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.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: He F, Wang W, Rutter WB, Akhunova A, Fernandez de Soto M, Szabo LJ, Jordan K, Rouse M, Akhunov E. Integrating Gene Expression Mapping, Epigenetics and GWAS to Understand the Genetic Control of Agronomic Traits in Hexaploid Wheat. Plant and Animal Genome XXVIII Conference, January 11-15, 2020.
• Type: Other Status: Published Year Published: 2020 Citation: Akhunov E. CRISPR-Cas-Based Precision Breeding in Wheat. Inari Company on-line seminar, April 6, 2020.
• 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. Genomic resources for studying the genetics of yield component traits in wheat. WheatCAP meeting, January 12, 2020.

Progress 11/15/18 to 11/14/19

Outputs
Target Audience: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. In addition, the project's objectives are aligned with the priorities of the Wheat CAP that includes an Industry Liaison Group, representatives in the National Wheat Improvement Committee, and breeders which coordinates research priorities with NAWG and Wheat Associates. PD Akhunov collaborated with The American Seed Trade Association (ASTA) to prepare video material about the application of gene editing to improve wheat. The video was broadly distributed to general public, academia and industry through the YouTube channel. Project results are also presented to wheat researchers, breeders and industry representatives at the International Wheat Yield Initiative meeting at Obregon, Mexico. The project team presented their discoveries to wheat researchers and breeders at the national and international conferences: 1) International Wheat Conference in Saskatoon (Canada), 2) Plant and Animal Genome (San Diego, CA), 3) National Association of Plant Breeders Meeting (Pine Mountain, GA), and 4) In Vitro Biology Meeting (Tampa, FL). The results of the project were disseminated to the international wheat community by publishing in the international peer-reviewed journals. An important aspect of preparing the next generation of plant breeders is the ability to communicate the sensitive issues of biotechnology and genome engineering to policy makers and the broader public. To accelerate the acceptance of genome editing tools for crop breeding, our project in collaboration with the Cornell Alliance for Science organized workshop (Speaking Science: CRISPR, Consumers and Conversations, Jan. 8-9, 2019, San-Diego CA) for postdoctoral researchers and PhD students. Workshop covered the basics of the CRISPR editing technology and strategies, and prepared graduate students and scientists to be effective science advocates that will have impact beyond the scope of this project. The KSU team also participated in the two monthREEU program run the KSU Department of Plant Pathology by hosting an undergraduate student. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In collaboration with the Cornell Alliance for Science, we have organized a workshop "Speaking Science: CRISPR, Consumers and Conversations", Jan. 8-9, 2019, San-Diego, CA. The course was attended by nearly 30 graduate students from the US wheat breeding programs. Postdoctoral researchers W. Wang and B. Tian made oral presentations about their projects at the national and international meetings, and also at a departmental seminar. B. Tian had a chance to participate in the KSU REEU program where he assissted co-PD Trick with superviding an undergradute student's research project. Project team shared their expertise in gene editing technologies with18 graduate students during the Genomic Technologies Workshop held at KSU June 17-21, 2019, and organized by the KSU Integrated Genomics Facility. How have the results been disseminated to communities of interest?The results generated by the project are presented to a broader public and private wheat research and breeding community through the publications in the peer-reviewed journals and presentations at the international and national meetings. Project team's work was featured in public media targeting broad audiences. PD Akhunov collaborated Kansas Wheat Commission and ASTA in creating movie titled "Plant Breeding and Innovation Part 3: Breeding for Better Bread" that was broadly distributed through the YouTube Channel. The current state of wheat genome editing initiatives at KSU have been presented to industry representatives and stakeholders through a series of presentations at the Kansas Wheat Innovation Center. Project directors organized workshops to train graduate students in gene editing technology and science communication skills. What do you plan to do during the next reporting period to accomplish the goals?Finalize phenotypic evaluation of wheat lines carrying several single-gene editing mutations, combinations of multiple genes edits and introgression of gene edits into the adapted germplasm. The phenotypic evaluations will be conducted in the field and greenhouse conditions in 2019-2020. Coming year will be also used to prepare manuscripts reporting the analysis of these gene-edited wheat lines.

Impacts
What was accomplished under these goals? Targeted yield component genes: We have created wheat lines (T1-T3 generations) carrying the CRISPR-Cas9-edited variants in the 10 homoeologous sets of genes (16 genes total) that have potential to affect yield component traits. These genes include TaCKX2-1, TaCKX2-2, TaGS3, TaTGW6-1, TaTGW6-2, TaTGW6-3, TaGW2, TaARF4, TaSPL16, TaGW7, TaAn-1, TaGSE5, TaLAC1-1, TaLAC1-2, TaGSK5, and TaSPL14. The phenotypic effects of gene editing were evaluated for TaGW2, TaGW7, TaAn-1, TaCKX2-1, TaLAC1-1 and TaGS3. Phenotypic effects of gene editing: By CRISPR editing of the TaGW7 gene, a homolog of rice GW7 encoding TONNEAU1-recruiting motif protein, we demonstrated that this gene controls grain weight and dimension traits in wheat (Wang et al., 2019). The edited copies of the TaGW7 gene had dosage-dependent effect on grain shape and weight with the double mutant showing stronger effects than the respective single copy mutants. The double gene knock-outs resulted in 2-3% increase in grain width and weight, and decreased grain length. Analysis of genome-wide patterns of SNP diversity in domesticated and wild wheat re-sequenced using exome capture showed that the TaGW7 gene homoeologs were the targets of domestication and lost diversity in cultivated wheat. CRISPR editing was used in this case to create novel diversity with potential to positively affect yield component traits. In rice, increase in GW7 gene expression was association with longer grain and higher quality. To investigate the effect of TaGW7 gene product increase on yield components in wheat, we created T1 transgenic plants overexpressing TaGW7 gene under the control of ubiquitin promoter. The editing of TaCKX2-1 gene encoding cytokinin oxidase in cultivar Bobwhite resulted in 10% increase in the number of grains per spike and grains per spikelet, without substantial negative impact on grain size and weight. In addition, we have generated gene edits in TaCKX2-1 of winter wheat cultivar Jagger and tetraploid spring durum wheat Kronos. The preliminary analyses of CRISPR-edited lines with TaGS3 and TaAn1 gene mutations showed ~7% increase and about 15% decrease in grain weight, respectively. For the latter gene, we have also created transgenic plants overexpressing the TaAn1 gene under the control of ubiquitin promoter. This transgene will allow for investigating the effect ofTaAn1 product increase on yield component traits. We did not observe substantial effect of TaLAC gene editing on grain size and weight traits. Further evaluations using larger populations are under way to accurately assess the phenotypic effects of CRISPR editing in these and other edited genes. Complexes of yield component genes: Additive interaction among genes controlling yield component traits was previously shown in rice and wheat, suggesting that by assembling optimal combinations of positively interacting alleles it is possible to achieve significant increase in yield potential. To investigate the effects of gene editing in several genes on yield component traits, we created a construct targeting 11 genes: TaGW2, TaGS3, TaGASR7, TaGW7, TaAn-1, TaCKX2 (1-3), TaTGW6, TaLAC, and TaGSE5. A total of 18 T0 plants carrying the CRISPR-Cas9 construct were regenerated and currently are being screened for the presence of editing events. In addition, in the progeny of crosses among lines carrying single-gene mutations in TaGW2, TaGASR7, TaAn-1, TaCKX2-1, and TaCKX2-2 we selected plants with different combinations of edited gene pairs. Phenotyping of the F3:4 generation double-gene mutant plants for yield component traits will be completed by the fall of 2020. To increase the multiplexing capacity of gene editing constructs, and to expand the number of possible targets in genome, we have established the Cpf1-based system for wheat. Cpf1 uses PAM sequence TTTV and requires shorter 40-44 bp-long CRISPR RNA (crRNA). In the wheat protoplasts, our Cpf1-based multiplex construct showed editing efficiencies higher than those exhibited by single-plex constructs and comparable to that of Cas9. We have used a Cpf1-based construct targeting a complex of gene TaGASR7-TaGW7-TaAn-1-TaSPL16-TaGW2-TaGS3-TaGSE5 to transform cv. Bobwhite and regenerated 57 T0 plants. The next-generation sequencing using our previously described amplicon pooling strategy revealed successful editing of several gene targets. Transfer of gene editing events to target cultivars: Previously, we have demonstrated that wheat lines expressing the CRISPR-Cas9 construct at high levels has the ability to induce novel gene editing events in the homologous gene copies of the F1 hybrids. We used this strategy to introduce mutations into the TaGW2 gene of multiple wheat cultivars. For five winter wheat lines from the US Great Plains and three CIMMYT high-biomass lines, we have obtained BC2F2:3 generation plants. The evaluation of TaGW2 gene transfer to the CIMMYT high-biomass line 3613474 showed 14.4% increase in thousand grain weight in greenhouse conditions. Evaluation of remaining lines will be conducted in 2020. Field phenotyping: We have performed field-based evaluation (Pullman, WA) of the spring wheat lines with the edited copies of the TaGW2 gene. Plants has been tested to be free from the CRISPR-Cas9 construct. In gene edited wheat lines with all three copies of the gene mutated, we found significant increase in thousand grain weight (15%) and grain protein content (8-10%), but no significant increase in grain yield. We have combined the CRISPR-edited TaGW2 gene with the edited variants of other yield component genes (e.g. CKX2) to investigate whether trade-off between yield-component traits can be removed to increase grain yield (see above). Workshops: As a part of Objective 5, in collaboration with the Cornell Alliance for Science, we have organized a workshop "Speaking Science: CRISPR, Consumers and Conversations", Jan. 8-9, 2019, San-Diego, CA. The course was organized alongside the Plant Animal Genome meeting and attended by nearly 30 graduate students from the US wheat breeding programs involved in Wheat CAP. The workshop was focused specifically on how to talk to audiences about gene editing, incorporating the communications and messaging data collected by the Center for Food Integrity's Gene Editing Coalition.In this course, participants reviewed the data on gene editing messages, talked about which analogies work, and which do not, and developed a consistent core message for broad audiences. Project participants helped to develop a training module on Gene Editing for the Genomic Technologies Workshop held at KSU June 17-21, 2019 by the KSU Integrated Genomics Facility. The workshop was attended by 18 graduate students.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wang W, Tian B, Pan Q, Chen Y, He F, Akhunova A, Evanega S, Yan L, Trick H, Akhunov E, CRISPR-Based Precision Breeding in Wheat, 1st International Wheat Conference, July 21-26, 2019, Saskatoon, Canada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E. Akhunov, Wheat Gene Editing Platform (WGEP) for improving Kansas wheat, Kansas Wheat Commission, 2019, March 28, 2019, Manhattan, KS
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: W. Wang, E. Akhunov, CRISPR-Cas9 genome editing technology as a tool for precision breeding in wheat. KSU Fall Seminar Series, 2019, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wang W, Tian B, Pan Q, Chen Y, He F, Akhunova A, Evanega S, Yan L, Trick H, Akhunov E, NIFA: CRISPR-Based Precision Breeding in Wheat. National Association of Plant Breeders Meeting, August 25-29, 2019, Pine Mountain, GA.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang W, Pan Q, Tian B, He F, Chen Y, Bai G, Akhunova A, Trick HN, Akhunov E. Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat. Plant J. 2019, 100(2):251-264, doi: 10.1111/tpj.14440.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Quanli Pan, Application of CRISPR/Cas9 to edit genes affecting seed morphology traits in wheat. M.S. Thesis, 2019, Kansas State University
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: W. Wang, Q. Pan, Y. Chen, B. Tian, F. He, A. Akhunova, S. D. Evanega, L. Yan, H. Trick, E. Akhunov Applications of Gene Editing to Improve Yield Component Traits in Wheat. In Vitro Biology Meeting, June 8-12, 2019, Tampa, Florida

Progress 11/15/17 to 11/14/18

Outputs
Target Audience: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. In addition, the project's objectives are aligned with the priorities of the Wheat CAP that includes an Industry Liaison Group, representatives in the National Wheat Improvement Committee, and breeders which coordinates research priorities with NAWG and Wheat Associates. The project team presents findings at the yearly Wheat CAP project meeting at the Plant and Animal Genome meeting. PD Akhunov collaborated with the KSU communication department to prepare video material about the project that was deposited to the KSRE YouTube video repository. The main findings of the project were highlighted in the press releases prepared by the KSRE communication department. Project results are also presented to wheat researchers, breeders and industry representatives at the International Wheat Yield Initiative meeting at John Innes Center, UK. The project results were presented to wheat researchers and breeders at the national and international meetings, such as Plant and Animal Genome. The results of the project were presented to the international wheat community by publishing in the international peer-reviewed journals. An important aspect of preparing the next generation of plant breeders is the ability to communicate the sensitive issues of biotechnology and genome engineering to policy makers and the broader public. To accelerate the acceptance of genome editing tools for crop breeding, our project develops educational modules for postdoctoral researchers and PhD students covering the basics of the CAS9 editing technology and strategies, and prepare graduate students and scientists to be effective science advocates that will have impact beyond the scope of this project. As part of this effort, our group in coordination with the Wheat CAP project organized a workshop at UC Davis, Kansas State University and Cornell University. During the workshop, co-PD S. Evanega covered various aspects of communicating the gene editing technology to public. PD Akhunov delivered a lecture and provided training in using the gene editing technology for functional validation of genes and generating beneficial allelic diversity for improving wheat. The project has recruited two Ph.D. students (Forrest C.C. Kan, Lei Lei), one M.S. student (Qianli Pan) and three postdoctoral researchers (Bin Tian, Yueying Chen and Wei Wang). The students and postdoctoral researchers attended the first USDA IWYP meeting organized in conjunction with the PAG meeting in San Diego. All graduate students and two postdoctoral researchers provided a training opportunity at the workshop organized jointly by our project and the Wheat CAP in UC Davis (Jan. 2018) and Kansas State University (July 2018). The workshop covered the basics of gene cloning and functional validation by CRISPR/CAS9 gene editing technology and provided training in communicating science to public. Three Ph.D. students, Forrest C.C. Kan, Xiaoyu Zhang, and Lei Lei have been trained and obtained skills and expertise in generation of gene editing plants using the CRISPR/CAS9 platform established in this project. Forrest and Xiaoyu have been trained in workshops as planned in the project. Changes/Problems:In the second year of the project, a postdoctoral researcher at KSU unexpectedly moved to a different university. The position has been advertised and will be filled in near future. This resulted in a slower than expected spending of available funds. This factor did not have any effect of scientific objectives that have been accomplished within the projected timeframe. None of project objectives was modified. What opportunities for training and professional development has the project provided?S. Evanega and the Alliance for Science team in collaboration with Akhunov group have conducted two primary training activities during the reporting year. In January, in coordination with the 2018 Wheat CAP workshop at the University of California Davis, our team organized a Gene Editing and science communication workshop. S. Evanega provided a two-hour primer on science communication, and why community engagement and science communication around editing is essential for its success. Akhunov provided a lecture on the basics of gene editing in wheat and hands-on training in bioinformatical tools used for designing gene editing experiments. This set the stage for a larger science communication workshop called "Speaking Science" that was held at Cornell University June 25-29th 2018. Our project supported ten WHEATCAP gradute studentsfrom eight US land grant institutions to attend the course. The course featured a series of lectures on science communications topics as well as several practical components, including engaging with tough journalists, sticking to message, and developing and delivering your personal science story to connect on shared values. Each participant ended the course having created an output in one of five tracks: how to write an op-ed, radio interview, video interview, print media interview, or social media strategy. Several of these pieces were aired or printed or are still under development. A number of students in the Op-ed track wrote opinion pieces on the USDA proposed bioengineered label, as that public comment period was open and on the cusp of closure. Example of participant output (print media track): https://www.kansasfarmer.com/crops/k-state-grad-student-finds-her-passion-study-wheat-genetics?NL=FP-008&Issue=FP-008_20180712_FP-008_220&sfvc4enews=42&cl=article_1_b&utm_rid=CPG02000000752256&utm_campaign=29336&utm_medium=email&elq2=665b75b943604237aae1f9eee0456529 Participants have also written about their experience in the workshop. One of the pieces was publlished on the AAAS website: https://www.aaas.org/blog/public-engagement-reflections/sharing-science-through-storytelling Following the workshop, participants offered feedback on the course in a focus group as well as through a post course survey. Students ranked exceptionally high the story telling element of the workshop as well as the mock interview with journalists. On a scale from 1 to 5 with 5 being most confident, participants on average cited an increase from 2.8 to 4.3 in their level of confidence when communicating to non-specialist audiences about their work. How have the results been disseminated to communities of interest?The results generated by the project are presented to a broader public and private wheat research and breeding community through the publications in the peer-reviewed journals and presentations at the international and national meetings. PDs prepared several press releases highlighting the discoveries made in the project. PD Akhunov collaborated with the KSU communication department to prepare video material about the project that was deposited to KSRE YouTube video repository. The current state of wheat genome editing initiatives at KSU have been presented to industry representatives and stakeholders through a series of presentations at the Kansas Wheat Innovation Center. Project directors organized three workshops at UC Davis, KSU and Cornell to train graduate students in gene editing technology and science communication skills. What do you plan to do during the next reporting period to accomplish the goals?Research: We have successfully accomplished Objectives 1 and 2 of the project by identifying 19 yield component genes, and developing and testing CRISPR/CAS9 constructs for their editing. As a part of Objective 3, in year 2019, we will continue developing transgenic lines for both spring and winter wheat to obtain the edited variants of all targeted yield component genes. All constructs designed and validated by the KSU groups will be also used by co-PD Yan's laboratory to develop gene-edited lines in winter wheat. As a part of Objective 4, all spring and winter wheat lines with edited genes will be grown in greenhouse conditions and phenotyped for yield component traits. These data will allow us to assess the transferability of gene information collected in other crop species into wheat, and simultaneously identify novel yield component genes that can be used for wheat improvement. The wheat lines showing significant positive changes in yield components will be selected for further phenotypic evaluation in both field and greenhouse conditions. These plants will also be used for crossing with the locally adapted Kansas and Oklahoma cultivars and CIMMYT high biomass and yield breeding lines. The CIMMYT lines with the edited gene variants will be shared with the IWYP hub in CIMMYT for further field-based evaluation and distribution among the IWYP partners. In the late fall of 2018 and spring of 2019, gene-edited spring wheat lines after removal of CRISPR/Cas9 constructs will be planted in the field. The phenotypic evaluation of these plants for yield component traits will be performed in spring/summer of 2019. The collected phenotypic data will allow us to assess the value of gene editing events for improving wheat yield in real-life agricultural setting. We currently discuss the possibility of growing some of the gene edited wheat lines in Arizona for collecting image-based phenological data using theLemnatec field Scanalyzer at Maricopa, AZ. In 2018, we showed that CRISPR/CAS9-based gene editing can be effectively used to modify genes in the F1 generation plants. In 2019, we will use this strategy for transferring gene editing events to multiple cultivars. Also, we will use the 707-1 transgenic line (high Cas9-expressing line) to establish more effective gene editing system for wheat. We expect that this modified strategy will help us to accelerate the development of gene-edited wheat lines by crossing cultivars with the transgenic wheat expressing a CRISPR/CAS9 construct. We will continue our experiments on simultaneous editing of multiple yield component genes in wheat to evaluate the effects of multiplex gene editing on phenotype. Additive interactions among genes controlling yield was shown for some genes in rice and wheat, suggesting that by combining positive alleles it is possible to achieve significant changes in yield components. A CAS9 multiplex editing provides the unique opportunity to test the effect combining multiple positive alleles on yield components by simultaneously editing multiple genes. Educational activities:As a part of Objective 5, in the third year of this project we will organize workshop focused specifically on how to talk to audiences about gene editing, incorporating the communications and messaging data being collected by the Center for Food Integrity's Gene Editing Coalition. This course is proposed for early January, 2019 alongside the Plant Animal Genome meeting to reduce travel costs. In this course we will review the data on gene editing messages, talk about which analogies work, and which do not. Participants will develop a consistent core message and further develop their outreach potential as early career genome editors.

Impacts
What was accomplished under these goals? Transgenerational activity of CRISPR-Cas9 facilitates multiplexed gene editing in wheat: During multiplex gene editing not all copies of genes in the polyploid genome can be simultaneously edited mostly due to low frequency of editing events in transgenic plants. By investigating the progenies of transgenic plans expressing multiplex gene editing constructs, we have shown that the targets not subjected to editing at earlier generations, can be edited in the next generations. The study reporting this finding was published in the inaugural issue of The CRISPR Journal (Wang et al., 2018). Increasing the efficiency of gene editing in wheat: One of the main limitations of CRISPR-CAS9 gene editing in wheat is its low efficiency ranging from 2% to 5% in most experiments. This low editing efficiency also limits our ability to transfer gene editing events to other cultivars by crossing the transgenic lines with the recurrent parental lines. Our team has identified transgenic Bobwhite line 707-1 expressing the CRISPR-CAS9 construct at a high level, four times higher than the level of the Actin gene expression. This line has been tested for the ability to induce novel gene editing events in the homologous gene copies of the F1 hybrids, which were created by crossing 707-1 line with CIMMYT high-biomass cultivars. By assessing the frequency of gene editing events using the the next-generation sequencing, we found that in the 707-1-derived F1 plants, nearly all homologous gene copies have been edited. In the same experiment, F1 plants derived from crosses with the Bobwhite transgenic lines 239-1 and 299-2 expressing CAS9 at much lower level showed no evidence of gene editing. This result suggests that the level of CAS9 construct expression can be one of the factors defining the frequency of gene editing events. Discovery of the 707-1 line provides unique opportunity to establish more effective gene editing system for wheat. Currently, we are using this CAS9-expressing line in the transformation experiments with constructs including only guide RNAs. We expect that this modified strategy will help us to accelerate the development of gene-edited wheat lines. Gene editing and EMS mutagenesis reveal inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat: The TaGW2 gene homoeologues have been reported to be negative regulators of grain size (GS) and thousand grain weight (TGW) in wheat. However, the contribution of each homoeologue to trait variation among different wheat cultivars was not well documented. We used the CRISPR-Cas9 system and TILLING to mutagenize each homoeologous gene copy in cultivars Bobwhite and Paragon, respectively. Plants carrying single-copy nonsense mutations in different genomes showed different levels of GS/TGW increase. In any combination, the double homoeologue mutants showed higher phenotypic effects than the respective single-genome mutants. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases of 16.3% (gene edited) and 20.7% (TILLING) in TGW. The highest single-genome increases in GS and TGW in Paragon and Bobwhite were obtained by mutations in the B and D genomes, respectively. These inter-cultivar differences in the phenotypic effects correlated with differences in the homoeologue expression levels. These results indicate that GS/TGW variation in wheat can be modulated by the dosage of homoeologous genes with inter-cultivar differences in the magnitude of the individual homoeologue effects. Generation of spring and winter wheat with the edited gene variants: In addition to the TaGW2 gene (Wang at al., 2018a; Wang et al., 2018b), we targeted 15 genes controlling yield component traits in wheat or other crops. This set of genes included homoeologous and paralogous copies of the TaCKX2, TaGS3, TaTGW6, TaGW2, TaGW7, TaAn-1, TaLAC, TaGASR7, TaGSE5, TaGRF4, TaSPL14, TaGL3, TaPAY1, and TaGW8 genes. CRISPR/CAS9 gene editing constructs validated for editing efficiency using the transient expression in the wheat protoplasts were used for the transformation of both spring and winter wheat cultivars. For spring wheat cultivar Bobwhite, we have obtained 344 transgenic plants positive for the presence of the Cas9 constructs targeting all 15 genes (on average, 20 plants/gene target). The next-generation sequencing (NGS) screening of 105 T0 plants identified plants that carried gene editing events in the TaLAC, TaAn-1, and TaGW7 genes. The NGS screening of remaining 239 plants is underway. The effects of TaGW7 gene edits on seed morphometric traits was confirmed in the T1 generation plants; the evaluation of T2 plants for this gene will be accomplished by the end of 2018. The phenotypic validation of TaLAC and TaAn-1 gene editing in greenhouse conditions will be finalized by the end of 2018. OSU group used 18 CRISPR/CAS9 constructs to transform 15,000 embryos collected from winter wheat including Duster, Smith's Gold, OK13209, Jagger, 2074, P15 and B1107. These constructs included 13 genes that were developed in KSU including TaAn-1, TaCKX2, TaGS3, TaTGW6, TaGW2, and five genes including TaBH1 and TaVRN1 that were recently identified to modify wheat yield components. At least six genes (TaAn-1, TaTGW6, TaGW2, TaBH1, TaVRN1, and TaVRS1) were found to be edited in T0 plants. The editing events were confirmed in the T1 progeny plants. The testing of T1 progeny of the plants carrying the edited TaBH1 gene showed decrease in plant height and increase in the number of spikelets. Transfer of gene-edited alleles to breeding lines: To transfer gene edited variants of the TaGW2 gene to other wheat cultivars, we have crossed TaGW2 gene triple mutants 239-1, which expresses a multiplex editing construct and homozygous for the loss-of-function mutations in all three copies of TaGW2, with 38 wheat cultivars. F1 plants have been genotyped by NGS to confirm heterozygosity and also to identify mutations on the homologous chromosomes of the recurrent parental line. The set of crossed lines includes 23 lines used in the Wheat CAP project, seven breeding lines from Kansas, and five high-biomass wheat lines from CIMMYT (Table 1). For some of these lines BC1F2 and BC2F1 generation seeds were obtained. The F1 generation plants produced from TaGW2 gene edited Bobwhite crosses with Colorado cultivars were shared with S. Haley for testing the effects of gene editing in Colorado environment.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang W, Simmonds J, Pan Q, Davidson D, He F, Battal A, Akhunova A, Trick HN, Uauy C, Akhunov E, Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat. Theor. Appl. Gen. 2018. https://doi.org/10.1007/s00122-018-3166-7.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang W, Pan Q, He F, Akhunova A, Chao S, Trick HN, Akhunov E, Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. The CRISPR Journal, 2018, 1, 65-74.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wang W, Pan Q, He F, Fernandez de Soto M, Ren J, Chen Y, Tian B, Akhunova A, Chao S, Trick HN, Akhunov E, Application of CRISPR/Cpf1-Based Genome Editing in Polyploid Wheat, Jan 13-17, 2018, San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pan Q, He F, Akhunova A, Chao S, Trick HN, Akhunov E. Application of CRISPR/Cas9 technology to analyzing gene function in the wheat genome and improving agronomic traits, Nebraska Plant Breeding Symposium, March 13th, 2018, University of Nebraska, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Wheat Gene Editing Platform (WGEP) for improving Kansas Wheat, KWC Research Committee meeting, March 21, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Wheat Improvement through Gene Editing, Flowers Foods and Grain Craft, June 6, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Gene Editing Opportunities and Capacities at KSU, Kansas Wheat Meeting, May 8, 2018, Manhattan, KS
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. CRISPR/Cas9 based wheat genome editing, Wheat CAP workshop, Jan 8th-12th, 2018 University of California, Davis, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Akhunov E. Editing the wheat genome, Sept. 19-21, 2018, International Conference From Seed to Pasta III, Bologna, Italy
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: L. Yan, W. Wang, H. Trick, Akhunov E. Gene editing for improving wheat yield component traits, 3rd Annual IWYP Program Conference, June 2018, John Innes Center, UK.
• Type: Other Status: Other Year Published: 2018 Citation: Akhunov E. Update on Gene Editing initiatives in wheat at KSU, Kansas Wheat Innovation Center meeting with TechAccel, Aug. 22, 2018, Kansas Wheat Innovation Center, Manhattan, KS

Progress 11/15/16 to 11/14/17

Outputs
Target Audience:PD Akhunov delivered presentations on the gene editing technology at the meetings with the Kansas Association of Wheat Growers, Kansas Wheat Commission (KWC), and Heartland Plant Innovations Center (HPI) andK-State Research and Extension. In addition, our project's objectives are aligned with the priorities of the Wheat CAP that includes an Industry Liaison Group, representatives in the National Wheat Improvement Committee, and breeders which coordinates research priorities with NAWG and Wheat Associates. The project team presents the results at the yearly Wheat CAP project meeting at the Plant and Animal Genome meeting. PD Akhunov presented wheat genome editing technology for the invited staffers supporting Kansas' congressional offices and selected citizen volunteer advocates. (September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA). Also, PD Akhunov presented and discussed the potential of gene editing technology for crop improvement at the Ag Innovation Forum organized by the Agricultural Business Council of Kansas City (June 14, 2017, Kansas City, KS). Project results are also presented to wheat researchers, breeders and indistry representatives at the Internatioan Wheat Yield Initiative meeting in CIMMYT, Obregon, Mexico. The project results were presented to wheat researchers and breeders at the national and international meetings, such as Plant and Animal Genome and CSSA-ASA-CSSA meetings. The results of the project were presented to the international wheat community by publishing in the international peer-reveiwed journals. An important aspect of preparing the next generation of plant breeders is the ability to communicate the sensitive issues of biotechnology and genome engineering to policy makers and the broader public. To accelerate the acceptance of genome editing tools for crop breeding, our project develops educational modules for postdoctoral researchers and PhD students covering the basics of the CAS9 editing technology and strategies, and prepare graduate students and scientists to be effective science advocates that will have impact beyond the scope of this project. As aprt of this effort, our group in coordination with the Wheat CAP project scheduled a workshop at UC Davis (Jan. 2018, 25 participants). During the workshop, co-PD S. Evanega will cover various aspects of communicating the gene editing technology to public. PD Akhunov will deliver a lecture and provide training in using the gene editing technology for functional validation of genes and generating beneficial allelic diversity for improving wheat. Changes/Problems:In the first year of the project, due to delays with hiring personnel there was a slower than expected spending during the first six months of the project. We have spent about 24.5% of funds. This factor did not have any effect of scientific objectives that have been accomplished within the projected timeframe. The project team had available personnel that started to develop many project objectives before this project was funded, that continued working on the same objectives. Now project teams are staffed with qualified researchers. None of project objectives of personnel were modified. What opportunities for training and professional development has the project provided?The project will train a new generation of breeders and geneticists in genome editing technology and the regulatory aspects of crop biotechnology thereby contributing to fulfilling the needs of industry, stakeholders, and academia in a trained workforce and making wheat production more profitable. S. Evanega and the Alliance for Science team have been engaging in meetings and dialogues at the nation and international levels with stakeholders who are examining public perceptions of gene editing technologies; the need to engage the publics on the technology, and determining best approaches for public engagement on gene editing. Since the proposal was developed there are some data available about the degree to which the public wants to be engaged on issues around gene editing. The data in food and agriculture are more limited than in medicine but more data are expected to be published between now and the end of 2017. In the months since the project was funded we have set up a robust communications monitoring portal where we are able to test messages about gene editing and track their impact over time. This portal was launched in beta form on August 11th, is currently in refinement and will be fully functional by September 15, 2017. We will use this monitoring portal to test messages that will be presented in the January 10th course. The effectiveness of the participants communicating those messages can be further tracked following the course. Our project in coordination with the Wheat CAP project scheduled a workshop at UC Davis (Jan. 2018, 25 participants). During the workshop, co-PD S. Evanega will cover various aspects of communicating gene editing technology to public. PD Akhunov will deliver a lecture and provide training in using the gene editing technology for functional validation of genes and generating beneficial allelic diversity for improving wheat. How have the results been disseminated to communities of interest?The results generated by the project are presented to a broader public and private wheat research and breeding community through the publications in the peer-reviewed journals and presentations at the international and national meetings. PD Akhunov presented wheat genome editing technology for the invited staffers supporting Kansas' congressional offices and selected citizen volunteer advocates (September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA). Also, PD Akhunov presented and discussed the potential of gene editing technology for crop improvement at the Ag Innovation Forum organized by the Agricultural Business Council of Kansas City (June 14, 2017, Kansas City, KS). The project results and material on gene editing are incorporated into the course material (Introduction to Systems Biology) developed at the Kansas State University. The gene editing plasmid constructs developed by the project are distributed to research collaborators. What do you plan to do during the next reporting period to accomplish the goals?Research: We have successfully accomplished Objectives 1 and 2 of the project by identifying 19 yield component genes, and developing and testing CRISPR/CAS9 constructs for their editing. As a part of Objective 3, in year 2018, we will continue developing transgenic lines for both spring and winter wheat to obtain the edited variants of all 19 yield component genes. All constructs designed and validated by the KSU groups will be also used by co-PD Yan's laboratory to develop gene-edited lines in winter wheat. As a part of Objective 4, all spring and winter wheat lines with edited genes will be grown in greenhouse conditions and phenotyped for yield component traits. These data will allow us to assess the transferability of gene information collected in other crop species into wheat, and simultaneously identify novel yield component genes that can be used for wheat improvement. The wheat lines showing significant positive changes in the yield components will be selected for further phenotypic evaluation in both field and greenhouse conditions. These plants will also be used for crossing with the locally adapted Kansas and Oklahoma cultivars and CIMMYT high biomass and yield breeding lines. The CIMMYT lines with the edited gene variants will be shared with the IWYP hub in CIMMYT for further field-based evaluation and distribution among the IWYP partners. In the fall of 2018, winter wheat lines carrying edited genes without CRISPR/CAS9 constructs will be planted in the field in Kansas and Oklahoma. The phenotypic evaluation of these plants for yield component traits will be performed in spring of 2019. The collected phenotypic data will allow us to assess the value of gene editing events for improving wheat yield in real-life agricultural setting. In 2018, we will study the efficiency CRISPR/CAS9-based gene drive for modifying genes by crossing cultivars with the transgenic wheat expressing a CRISPR/CAS9 construct. For this purpose, we will analyze F1 lines by next-generation sequencing for the presence of novel editing events. If CRISPR/CAS9-based gene drive system will be effective in wheat, it will allow for easy and time-effective transfer of favorable gene edits into any cultivar. We will start evaluating the effect of simultaneous editing of multiple yield component genes on phenotype. Additive interactions among genes controlling yield was shown for some genes in rice and wheat, suggesting that by combining positive alleles it is possible to achieve significant changes in yield components. A CAS9 multiplex editing provides the unique opportunity to test the effect combining multiple positive alleles on yield components by simultaneously editing multiple genes. For this purpose, three single polycistronic constructs including sgRNAs targeting three sets of genes, that are shown to act additively and affect yield components, will be assembled from the multiple gRNA-tRNA units and used for wheat transformation. Educational activities:As a part of Objective 5, in the second year of this project we will conduct our first introduction to communicating about gene editing at UC Davis workshop (25 participants) in Jan. 2018. Following that introductory course, we will offer a two-day storytelling workshop to help participants refine their personal story and message. We will find opportunities for participants of the communications workshop to place blog posts, speak at conferences about their experience communicating about editing (at CRISPRcon, for example), and record their personal stories in the form of pod casts. In addition, in 2018 we will be fully monitoring the global conversations about gene editing in wheat and food and ag more broadly and will have concrete data on the state of the public sentiment and media coverage of this technology.

Impacts
What was accomplished under these goals? Genetic studies in cereal crops, fueled by the availability of sequenced genomes, and by the advances in sequencing technologies, identified a number of genes that control major agronomic traits including yield components. The recent release of the wheat genome sequence now providesopportunity to easily extrapolate gene mapping information from other crops to wheat. CRISPR/CAS9-based genome editing has emerged as a disruptive technology that can take full advantage of the available genomic information and help to rapidly transfer traits into wheat cultivars. The CRISPR/CAS9 acts as a pair of scissors to cut DNA and introduce changes into the genetic code of specific genes. Our project explores the capabilities of this novel CAS9-based gene editing technology to unlock the potential of the complex wheat genome, thereby building a foundation for transformative approaches to wheat improvement. Wheat-optimized CRISPR-CAS9 system for multiplexed gene editing: We have optimized CAS9 gene editing platform for effective gene editing in wheat as well as for simultaneous targeting of multiple genomic regions. Our wheat gene editing platform includes: 1) wheat codon-optimized CAS9 enzyme; 2) procedure for high-throughput screening of designed gRNAs for their editing efficiency using the wheat protoplast assays; 3) procedures for the next-generation sequencing of multiplexed amplicons and bioinformatical analysis for the quick assessment of the frequency and types of editing events in the wheat genome; 4) simplex and multiplex (tRNA-based spacer separated) gene editing constructs based on Golden Gate assembly strategy. CRISPR/CAS9 gene editing constructs:The developed CRISPR/CAS9 pipeline was used to perform the quick functional screening of nearly 400 gRNA constructs. For 14 genes controlling yield components in wheat or other crops, CRISPR/CAS9 gene editing constructs were designed and successfully evaluated for editing efficiency using the transient expression in the wheat protoplasts followed by next-generation sequencing. Both types of constructs targeting a single gene and multiple genes were designed. The latter includes a construct targeting 10 genes that act as suppressors of yield component phenotypes in wheat and other crops. Generation of spring and winter wheat lines with the edited gene variants: A total of 13 gene editing constructs were used for transforming wheat cultivar Bobwhite applying the biolistic transformation approach in the laboratory of co-PD H. Trick. For 12 constructs, at least 10 T0 plants were regenerated. The transformation experiments with the remaining constructs are at the different stages of completion. By next-generation sequencing of the produced T0 plants we have confirmed the presence of editing events in the targeted genomic regions of five genes. Additonal transformants are being generated to obtain editign events in the remainign genes. Co-PD Yan's group established effective transformation protocol for winter wheat. The TaGW2 gene editing construct created at KSU was used to transform two winter wheat lines B1107 and P15 from Oklahoma. For each line, three transgenic plants were identified that have indels in the TaGW2 gene. The same TaGW2 construct has been delivered into the calli of cultivar 'Duster', a locally adapted Oklahoma winter wheat cultivar. The regeneration of transformed plants is currently underway. Assessing the phenotypic effects of CRISPR/CAS9-induced mutations: For the TaGW2 gene editing construct, 15 T0 plants carrying mutations in the TaGW2 gene were regenerated. A total of 59 T1 families that were derived from these T0 lines carried heterozygous or homozygous mutations in one, two or all three homoeologous copies of the TaGW2 gene. The T1 families from T0 lines that carry knock-out mutations in all three homoeologous copies of the TaGW2 gene (genotype aabbdd), and T1 families that carry homozygous mutations in either the A and D genomes (genotypes aaBBDD or AABBdd) were used for digital phenotyping using the Marvin seed analyzer. Non-mutagenized cultivar Bobwhite (genotype AABBDD) was used as a control. The effect of mutagenizing all three TaGW2 gene copies had more substantial effect on phenotype than that obtained by mutagenizing a copy of the gene in only one of the wheat genomes. Compared to the wild type plants, the aabbdd genotypes had the thousand-grain weight (TGW), grain area (GA), grain width (GW), and grain length (GL) increased by 20%, 10%, 9% and 2%, respectively. The edited plants with the AABBdd genotypes showed 7%, 2%, 3% and 1% increase in the TGW, GA, GW, and GL, respectively. These results suggest that the homoeologs of the TaGW2 gene has a dosage-dependent effect on seed size phenotypes, and indicate that the CAS9's ability to introduce changes to any or all three wheat genomes simultaneously provides a powerful tool for analyzing gene function in the polyploid genome complementing TILLING populations. Transfer of gene-edited alleles to breeding lines: A total of 35 spring and winter wheat varieties or breeding lines were crossed with the edited gw2 genotype carrying mutations in all three genomic copies of the TaGW2 gene. For Thatcher x gw2 cross, 10 F1 plants were genotyped to confirm the presence of the TaGW2 mutations and 600 F2 seeds have been planted.The seed dimension phenotyping of Thatcher/gw2 F1 lines showed the mid-parent values indicating that the gw2 mutant alleles have an additive effect on phenotype. A total of 151 F1 plants were obtained by crossing seven locally adapted Kansas breeding lines with the gw2 mutant, 57 F1 plants were generated by crossing this mutant with 4 CIMMYT high biomass lines, and 293 F1 plants were derived from crosses of 23 US wheat breeding lines from the Wheat CAP project with the gw2 mutant. The F1 lines will be backcrossed to the recurrent parents in 2017-2018 season. These results demonstrated the feasibility of improving wheat yield components by introducing CRISPR/CAS9-generated novel allelic variation into the wheat cultivars.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu M, Lei L, Miao F, Powers C, Zhang X, Deng J, Tadege M, Carver BF, Yan L. 2017. The STENOFOLIA gene from Medicago alters leaf width, flowering time and chlorophyll content in transgenic wheat. Plant Biotechnol J. DOI: 10.1111/pbi.12759.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, W., Akhunova, A., Chao, S. & Akhunov, E. Optimizing multiplex CRISPR/CAS9Cas9 system for wheat genome editing. biorxiv, 2016, https://doi.org/10.1101/051342
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Liu M, Lei L, Miao F, Powers C, Zhang X, Deng J, Tadege M, Carver BF, Yan L. 2017. The dicot Medicago gene STF increased leaf width in monocot wheat. 13th International Wheat Genetics Symposium, April, 2017. Tullin, Austria.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, Pan Q, He F, Akhunova A, Evanega SD, Yan L, Trick H, Akhunov E. Genome editing for improving wheat yield and yield-related traits. 2nd IWYP Conference, 20-23 March 2017 2017, Obregon, Mexico.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, Pan Q, He F, Akhunova A, Evanega SD, Yan L, Trick H, Akhunov E. Genome editing for improving wheat yield and yield-related traits. USDA PD meeting and NAPB, August 7-8 2017, UC Davis, Davis, CA, USA
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: E. Akhunov. Application of Multiplex CRISPR/Cas9-Based Genome Editing Strategy for Targeting Multiple Agronomic Genes in Wheat. 2017, ASA, CSSA, and SSSA Annual Meeting in Tampa, FL.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: E. Akhunov. Wheat genome editing. Presentation for the invited staffers supporting Kansas congressional offices and selected citizen volunteer advocates. September, 2017, Kansas Wheat Innovation Center, Manhattan, KS, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Akhunov E. The Potential for Gene-editing in Cereal Crops, Ag Innovation Forum, The Agricultural Business Council of Kansas City. June 14, 2017, Kansas City, KS.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang W, He F, Pan Q, Chao S, Trick HN, Akhunova A, Akhunov E. Application of Multiplex CRISPR/Cas9-Based Genome Editing Strategy for Targeting Multiple Agronomic Genes in Wheat. Plant and Animal Genome meeting, Jan 14-18, 2017, San Diego, CA, USA