Progress 12/01/16 to 11/30/17
Target Audience:1) Geneticists, genome biologists and scientific community at large. 2) Wheat geneticists and breeders. 3) Wheat growers. Changes/Problems:
What opportunities for training and professional development has the project provided?Drs. Lei Hua and Zhengzhi Zhang, originally worked on other projects of Li Lab and Yang Lab in South Dakota State University (SDSU) and Iowa State University (ISU), respectively. They were reassigned to work on this project, but they were supported by other funds as in-kind contribution during the earlier stage of the project. Lei Hua, previously trained in rice biology and soybean physiology, is working on designing and testing genome-specific primers and sequencing GS genes from FDR. Zhengzhi Zhang, previously trained in genome mapping, is working on establishing CRISPR/Cas9 system and constructing GS-specific CRISPR/Cas9 constructs for Agrobacterium-mediated wheat transformation. More recently, a third postdoc, Dr. Dangping Luo, joined this project in Yang Lab for characterizing transgenic plant and developing the protocol for mutation detection. At SDSU, MS student Samantha Shaw and undergraduate student Melissa Kerr were trained by the project in the past year using other funds as in-kind contributions. Both worked on PCR amplification of TGW6 genes from tetraploid landraces, sequencing the PCR products and analysis of the sequences for beneficial mutant alleles. These side projects identified several nonsense mutations in this negative GS regulator. Drs. Lei Hua and Zhengzhi Zhang will continue to work on this project in the coming year. Zhengzhi Zhang will work on Objective 1 to construct new CRISPR vectors carrying two copies of Cas9 and test their editing efficiency. Lei Hua will work on Objective 2 to screen the T0 and T1 transgenic population for beneficial mutation and on Objective 4 to screen the backcrossing populations using edit-specific markers. We will recruit a Ph.D. student at SDSU to work on Objective 3 to evaluate phenotype effect of the mutations. How have the results been disseminated to communities of interest?A. Journal Publications & Conference presentations The results have been disseminated through a journal publication and a conference presentation. B. Scientific Resources Generated We have deposited sequences of 55 GS genes or gene fragments from FDR in the GenBank and posted sequences of these 55 GS genes and the 56 pairs of gene-specific primers on the project webpage (https://wheatgenomics-sdsu.github.io/). In addition to designing guide RNA genes and detecting edit mutations, these primers also can be used for mining GS gene alleles in landraces and wild relatives of wheat. C. Community Resources Generated Dr. Bing Yang gave three lectures (1. CRISPR/Cas9 technologies for plants; 2. Hands-on experience: CRISPR guide RNA design; and 3. Hands-on experience: CRISPR/Cas9 construction) this summer in 2017 Workshop on Plant Genome Editing, organized by the Research Promotion Center of Life Sciences, Ministry of Science and Technology of Taiwan, July 10, 2017. Approximately 100 scientists and students participated in the workshop. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Develop an improved CRISPR/Cas9 system for editing GS candidate genes We expect to accomplish the development of the CRISPR constructs in the coming year as proposed (Project Timetable). According to recent publications, CRISPR editing rate in wheat is less than 5%. Considering that the low edit mutation rate is a bottleneck to the application of CRISPR genome editing to wheat improvement, we will prioritize our research under this Objective in following aspects to maximize the success of the project. Increase abundance of edit-enabling Cas9/sgRNA ribonucleoprotein complex by doubling expression levels of the Cas9w and sgRNA genes. We hypothesize that the low edit rate in wheat is associated with its large genome size, and increasing content of Cas9 enzyme and sgRNA in the nucleus may be a solution. As we already developed two sgRNA to target each GS gene, we will deploy two copies of Cas9w in the binary vector pCL-Cas9w, one under ZmUbi promoter from maize and another under OsUbi promoter from rice. Identify the most active sgRNA genes for stable wheat transformation by using protoplast system and deep sequencing. Hypothesizing that different sgRNAs target different regions of the gene of interest with different efficiencies, we will design several sgRNA genes to target a GS gene. A GFP reporter will also be included in the construct, which will be transformed into wheat protoplast. The cells expressing GFP will be sorted and used for DNA isolation. Gene-specific PCR products, including the sgRNA targets, will be sequenced by an Illumina DNA analyzer, the reads will be mapped to the target region, and edit rate will be estimated. The sgRNA genes showing the highest edit rate will be used to construct the single and complex targeting CRISPR vectors for Agrobacterium-mediated transformation. Increase number of T0 transgenic plants. This is another approach to ensure production of mutations for each construct. Based on the close collaboration with UCDPTF, we will regenerate T0 transgenic plants from all transformed calluses. Understand the effect of genome complexity (genome size and ploidy) on CRISPR mutagenesis efficiency. We will test a set of gene loci in wheat species of different ploidy levels (diploid, tetraploid and hexaploid) using protoplast assay and deep sequencing. This will provide insights into how the genome size and ploidy affect the CRISPR efficiency and lay a foundation for developing strategies to improve wheat genome editing efficiency. Objective 2. Identify mutations in the GS candidate genes We will screen the T0 and T1 transgenic populations for beneficial mutations and transgenes as follows: Genotype T0 and T1 transgenic plants for mutations of target genes through PCR amplification of relevant regions and Sanger sequencing of PCR-amplicons. Identify mutant T1 progeny plants containing desired mutations but no CRISPR constructs using a PCR approach. We will develop STARP or KASP markers for the mutant alleles. We will cross the plant carrying beneficial mutations with elite wheat cultivars from South Dakota State University and IWYP hub. This will accelerate transferring the edit mutations into breeding populations. We will also intercross the transgenic plants carrying the same transgenes but derived from different transformation events even if they do not carry edit mutations. From the progenies of these crosses, we expect to select plants carrying four doses of transgenes because the transgenes are most probably located on different chromosomes or chromosome arms, and stacking of transgenes is expected to increase editing rate. Objective 3. Characterize the effect of mutations on GS and GN In the coming year, this part of the research will focus on phenotypic characterization of GS and GN mutants (Objective 3.1). The homozygous mutants will be measured along with wild type FDR for GS and GN, and statistic tests will be used to examine the effect of the edit mutations on these traits. This research activity will help identify beneficial mutant alleles. Objective 4: Transfer beneficial mutations into elite durum wheat We will cross the GS/GN mutants identified in FDR with the elite durum cultivar Divide and backcross the hybrids to Divide. The backcrossing progenies will be screened for the GS/GN mutations. A barrier to gene introgression between hexaploid and tetraploid wheat is hybrid necrosis due to the interaction between the Ne1 gene on chromosome arm 5BL of tetraploid wheat and the Ne2 gene on 2BS of hexaploid wheat. We have crossed FDR and Divide. Their F1 hybrids are growing in a greenhouse and currently at the 3-leaf stage, and have not shown necrosis phenotype, suggesting a good possibility that the GS/GN mutations developed in FDR can be transferred into durum.
What was accomplished under these goals?
Objective 1: Develop an improved CRISPR/Cas system for editing the GS candidate genes (20%) Objective 1.1. Develop an improved CRISPR/Cas9 system for wheat genome editing To develop an improved CRISPR/Cas9 system seamlessly compatible with the high-efficiency wheat transformation procedure, we inserted Gateway recombination sites attR1 and attR2 and a wheat codon-optimized Cas9 gene Cas9w in the T-DNA region in the wheat transformation vector pLC41. This vector was provided by the University of California Davis Plant Transformation Facility (UCDPTC). Through this progress, we generated a Cas9 destination expression vector pLC-Cas9w. In this binary vector, the Gateway recombination sites are used to accept a single guide RNA (sgRNA) expression cassette, and theCas9w gene is driven by ZmUbi promoter for over-expression. At the same time, we have built two platforms to construct guide RNA genes: one expressing two guide RNAs to target single genes and another expressing up to eight guide RNAs to target multiplex genes for mutagenesis. The sgRNA cassette was first constructed in a guide RNA vector and flanked by Gateway recombination sites attL1 and attL2 and can be readily mobilized into pCL-Cas9w by recombination, resulting in a construct expressing both Cas9w and guide RNA genes for wheat transformation. This CRISPR system has been tested in a wheat mesophyll protoplast system using a reporter gene targeting system, in which a GFP mutant containing a 1-bp insertion at its 5' region (GFP+1), which causes a frame shift and loss of the GFP function, is driven by 35S promoter, and a guide RNA gene targets the mutation site for recovering the GFP function. Detection of GFP signal in wheat protoplast indicated that this CRISPR system can be used for wheat gene editing. Objective 1.2. Construct CRISPR/Cas9 plasmids targeting the grains size candidate genes We identified 45 grain size (GS) candidate loci in the wheat genome based on similarity of protein sequences and expression patterns to the GS genes in the model plants. In silico mapping of these GS loci in the diploid wheat and barley genomes showed that (1) several gene families have been amplified in the wheat lineage, (2) a significant number of the GS genes are located in the proximal regions surrounding the centromeres, and (3) more than half of candidate genes are negative regulators, or their expression negatively related by microRNAs. Identification and genome mapping of the wheat GS gene homologs will not only facilitate candidate gene analysisbut also open the door to improving wheat yield using reverse genetics approaches by mining desired alleles in landraces and wild ancestors and to developing novel germplasm by TILLING and genome editing technologies. This result was recently published in journal Theoretical and Applied Genetics. From these 45 candidate genes, we selected 16 negative GS genes and four microRNA-regulated positive GS genes as genome editing targets. Common wheat is a hexaploid species containing A, B and D genomes, and homoeologous genes from these genomes show ~97% similarity. Therefore, development of genome-specific PCR primers is a key to identify CRISPR mutations in individual homoeologous genes in polyploid wheat. We have designed 153 pairs of PCR primers, of which 56 generated single bands for 20 genes in cultivar Chinese Spring (CS) and 55 pairs of primers worked in cultivar Fielder (FDR) cultivars.Because guide RNAs are usually designed to target the 5' part of a gene for best knockout effect, these primers were designed intentionally to amplify the 5' portion of the negative GS genes or the microRNA recognition sites of the four microRNA-regulated positive GS genes. Variation in genic sequences exists among wheat cultivars, but 100% sequence homology is required for CRISPR targeting. The reference sequences of the wheat genome were obtained from cultivar CS, but the CRISPR/Cas9 constructs will be transformed in cultivar FDR. To design guide RNAs for editing GS genes, we sequenced the grain regulatory genes from FDR. Sequencing of 55 PCR products generated ~48 kb sequences and identified 57 SNPs and eight indels, of which 37 SNPs found in exons, showing 99.86% identity as compared to CS. All the FDR sequences for the 55 fragments of 20 orthologous GS loci have been forwarded from Li Lab to Yang Lab for designing guide RNAs. We have deposited the FDR gene sequences in GenBank and posted the sequences of the GS genes from FDR and homoeolog-specific primers on the project webpage. Using the CRISPR Genome Analysis Tool (CGAT) program, we designed 40 guide RNA genes. For each GS gene, two guide RNA genes were designed to target the exonic sequences conserved (identical) among its A-, B- and D-genome homoeologs. For single targeting of a GS gene, the two gene-specific sgRNA genes under wheat U6.1 promoter are synthesized; for multiplex targeting of up to four GS genes, a cassette expressing up to eight guide RNAs is constructed based on the tRNA-sgRNA architecture. The either sgRNA cassette was cloned into a guide RNA vector, flanked by attL1 and attL2, and mobilized into the binary vector pCL-Cas9w for Agrobacterium-mediated wheat transformation. We have generated 13 constructs, and construction of another three single-gene constructs and 6 multiple-gene constructs is under way. 1.3. Transform CRISPR constructs into wheat by Agrobacterium-mediation We have transformed the 13 constructs into Agrobacterium tumefaciens strain EH105 and submitted to UCDPTC for transformationinto FDR wheat. We recently received 27 T0 transgenic plants for the five constructs of the first two submissions. Currently, we have developed close collaboration with David Tricoli, the UCDPTF manager, in which the UCDPTF will coauthor the publication from this project. This will be critical for reducing the experimental cycle time, maximizing mutation rate by modifying experimental protocol and generating more transgenic plants without cost increase. Objective 2: Identify mutations in the GS and GN candidate genes (1%) We are currently characterizing the T0 transgenic plants for thepresence of transgenes (Cas9 and sgRNAs) and expression of both Cas9 and gRNA genes, as well as the presence and nature of expected mutations at the target sites. Objective 3: Characterize the effect of mutations on GS and GN (0%) No research activity wasplannedunder this objective in this period of the project Objective 4: Transfer beneficial mutations into elite durum wheat (0%) No research activity wasplanned underthis objective in this period of the project
Li W, Yang B. 2017. Translational genomics of grain size regulation in wheat. Theoretical Applied Genetics. 130:17651771.