Source: KANSAS STATE UNIV submitted to
GENOME EDITING FOR IMPROVING WHEAT YIELD AND YIELD-RELATED TRAITS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1011365
Grant No.
2017-67007-25932
Project No.
KS1011667
Proposal No.
2016-06711
Multistate No.
(N/A)
Program Code
A1142
Project Start Date
Nov 15, 2016
Project End Date
Nov 14, 2018
Grant Year
2018
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
20115491080100%
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 11/14/17

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