Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
CRISPR-BASED GENOME EDITING OF GRAIN SIZE REGULATORS FOR NOVEL VARIATION TO INCREASE WHEAT GENETIC YIELD POTENTIAL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1011261
Grant No.
2017-67008-25934
Project No.
SD00G637-16
Proposal No.
2016-06712
Multistate No.
(N/A)
Program Code
A1142
Project Start Date
Dec 1, 2016
Project End Date
Nov 30, 2019
Grant Year
2019
Project Director
Li, W.
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Grain yield is one of most important targets for wheat breeding. While wheat yield isimpacted by climate change, the rate of wheat genetic yield gain, which is mainly driven by increase in grain number (GN) per unit area of land, has slowed down. Under such a backdrop, the International Wheat Yield Partnership (IWYP) was set up with a common goal to increase wheat yields by 50% by 2034. To meet this demand, annual wheat yield increases must grow from the current level of below 1% to at least 1.7%. This quantum increase in genetic yield will require the development of breakthrough approaches for wheat improvement. This project seeks to increase wheat yield potential through precise modification of the genetic factors (genes) controlling wheat grain size (GS) or grain weight (GW), which arebelieved to be major drivers for further yield growth, using the most updated genome-editing technology called CRISPR/Cas9. The research team will first improve the efficiency of the CRISPR/Cas9 system in wheat, integrate it into the wheat transformation vector, and develop 30 constructs to knock out 20 GS/GN genes for novel variation. These 30 constructs will be used to generate 150 transgenic wheat lines. Every tiller of the transgenic plants will be screened for desired mutations using sensitive molecular assays, and GS traits will be measured using a computer image-based high throughput approach. Eventually the mutants showing increased GS will be selected, and knowledge of GS genetics will be gained. Compared to traditional genetic engineering, a significant advantage of the genome-editing technologies is that their end products, the knockout mutants, can be non-GMO. Therefore, the useful mutants developed from this project can be used in wheat breeding as novel germplasm. Finally, some of the useful mutations will be transferred into durum wheat to improve its yield potential. Thus, this project is expected to significantly contribute to a more profitable wheat industry, US rural economy and the IWYP's goal.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011549104040%
2011549108045%
2021549108015%
Goals / Objectives
The goal of this project is to develop an improved CRISPR/Cas system for editing grain size (GS) regulators to create novel variation that leads to significant increases in the genetic yield potential of wheat, which is consistent with the common goal of the International Wheat Yield Partnership (IWYP)to increase wheat yield by 50% by 2034.Objectives of this project include: (1) to develop an improved CRISPR/Cas9 system for editing GS candidate genes; (2) to identify mutations in the GS candidate genes; (3) to characterize the effect of mutations on GS and GN; and (4) to transfer beneficial mutations into elite durum wheat.
Project Methods
Methods for Objective 1: Develop an improved CRISPR/Cas system for editing the GS candidate genesThe research team has optimized Cas9 based on wheat codon usage (Cas9w) and identified wheat U6.1 and U6.3 promoters that are most effective in driving sgRNA genes in wheat cells. These components will be integrated into the wheat transformation vector pLC41, which is used in an improved wheat transformation procedure and provided by the University of California Davis Plant Transformation Facility(UCDPTF) under a service contract, to develop the first-generation CRISPR/Cas system for wheat. The initial vector PCL-Cas9w will be able to clone 4 sgRNA genes driven by individual U6.1 or U6.3 promoters. An alternative CRISPR/Cas9w system in wheat, which is based on the tRNA-gRNA architecture for expression of up to 8 sgRNA genes will also be adopted and tested.The second step in improving the wheat CRISPR/Cas system is to reduce the off-target effect and to expand target range. Recent studies in animals showed that engineering Cas9 protein with PAM-interacting domain by D1135V, R1335Q and T1337R substitutions and the RuvC domain by K848A, K1003A and R1060A substitutions significantly expanded range of targeting sequences and improved targeting specificity of Cas9 protein, respectively. These mutations will be integrated into the wheat codon-optimized Cas9w through a gene synthesis service, and their efficacy will be tested for eight guide RNA sequences selected from the homoeologs of single-copy GS candidate genes such as DEP1 and LP. Once progress is made, the wheat gene editing platform will be upgraded accordingly.Homoeolog-specific primers will be designed using aweb tool GSP (Genome-Specific Primers in polyploid species; http://probes.pw.usda.gov/GSP), validated by using nulli-tetrasomic stocks, and then used to amplify the GS and GN candidate gene sequences from Fielder wheat. Target sites will be selected from the conserved sequences in 5' region of the 16 negative GS candidate genes and the microRNA recognition sites of the 4 GS positive genes regulated by microRNAs: GLW7, GS2, GW8, and IPA1. A total of 30 constructs will be developed, each will carry 4 - 6 sgRNA genes. Expression efficiency of Cas9w and sgRNA genes in each construct will be confirmed in wheat protoplasts by reverse transcription quantitative PCR (RT-qPCR), and editing efficiency will be confirmed by T7 enzyme 1 (T7E1) assays, which recognize and cleave non-perfectly matched DNA, before the constructs are submitted to University of California Davis Plant Transformation Facility (UCDPTF) for stable Agrobacterium-mediated transformation into "Fielder" wheat to produce 5 transgenic lines for each construct, 150 transgenic plants for 30 constructs. The top 2 - 3 of each category (a total of 8 - 12 genes) as the group 1 will be prioritized for mutagenesis and in-depth characterization. At a later stage with the improved CRISPR system, the remaining 8 - 12 candidate genes as group 2 will be studied with relatively small budget and effort.Methods for Objective 2: Identify mutations in the GS and GN candidate genesFor cost-effectiveness, an early and rapid mutation detection by PCR T7E1 assays, and subsequently by Sanger sequencing will be performed to identify edit-positive callus lines, from which transgenic plantlets will be regenerated. Given each transformation construct contains an average of five sgRNA genes and an average of 10 callus lines for each construct are screened for three homoeologous copies of each candidate gene, a total of 4,500 (30 x 10 x 5 x 3) T7E1 assays will be conducted. Each tiller of T0 transgenic plants will be screened by T7E1 assays using the homoeolog-specific primers. Given 3 tillers per plant are screened for three homoeologous copies of each candidate gene, a total of 6,750 (150 x 5 x 3 x 3) T7E1 assays will be performed. PCR products from the positive T0 transgenic tillers will be sequenced by the Sanger method. The frequencies of all types of mutations will be estimated, and mutation genotypes in all three homoeologs will be determined. Spikes from the edit-positive tillers of the T0 plants will be harvested individually, cataloged and advanced to T1 generation. Meanwhile, a diagnostic marker will be developed by targeting the mutations to facilitate high-throughput mutation selection and future application in molecular breeding.For evaluating the contribution of individual candidate genes and individual homoeologs to the GS and/or GN phenotype, T0 or T1 plants carrying multiple mutations will be backcrossed to wild type Fielder, and the BC1F1 and BC1F2 populations will be screened by diagnostic markers.Methods for Objective 3: Characterize the effect of mutations on GS and GNThe mutants will be grown in greenhouse along with wild type Fielder for each batch, and four individuals (biological replicates) will be planted for statistical analysis. In addition to GS, other yield-related traits will also be scored or measured, including grain number per spike (GN/S), grain number per spike (GN/P), TGW, spike length (SL), spikelet number (SN), date to heading (DTH), plant height (PH), and tiller numbers (TN). Spikes from each plant will be harvested individually and threshed manually, and seeds of each spike will be cleaned, bagged and dried before measured. Grains from a spike will be counted and weighted, GN/S, GN/P and TGW will be estimated. For GS measurement, seeds from a spike will be spread onto the glass surface of a HP Scanjet G3110 scanner (HP Inc., Palo Alto, CA), covered with a black cloth and scanned at 300 dpi with color format set as grayscale. The images will be saved in the JPG format and fed into GrainScan software for batch processing. Mutation effect of a single homoeologous gene locus on GS, TGW, GN/S, GN/P, SPL, SPN, PH and DTH will be determined by the t-tests using the wild type Fielder as the control. Differences among the wild type, single, double and multiple mutations will be examined by two-tailed analysis of variance (ANOVA) with the cut-off value set as 0.05. The mutants showing significant phenotype effect on GS or/and GN will be advanced for further characterization.Once GS and GN mutants are developed, they will be crossed and double mutants will be selected for exploring new possibilities to break the negative correlation (trade-off) between these two important traits. The GS and GN phenotypes will be compared between the multiple mutations and the cognate single mutations for additive and/or epistatic effect between the genes involved by two-tailed ANOVA. For example, the mutations of TaGW2, TaTGW6 and TaCKx will be combined because all these genes are negatively associated with GS, TGW and potentially GN.The GS and GN mutants will be characterized at molecular level. For the transcription factor, transcription of their downstream genes, such as DEP1, SRS5, GW7, and TaCKx homologs will be examined RT-qPCR, for the 4 microRNA-regulated genes, expression of themselves will be measured. In addition, expression of genes required for cell proliferation, such as histone H1, transcription factor E2F2 and cyclin-T1, and for cell expansion, such as TaExpA6 will also examined.Methods for Objective 4: Transfer beneficial mutations into elite durum wheatDurum wheat and common wheat sharethe A and B genomes and are ready to cross,to produce partially fertile F1 hybrids. The T0 plants enriched for the desired mutations in the A or/and B genome will be sorted out and crossed and backcrossedwith the elite US durum wheat cultivar "Divide". The BC populations will be screened by mutation diagnostic markers for mutant alleles and to eliminate the D-genome chromosomes and transgenes. The phenotypic effect of the mutant alleles will be evaluated on six individuals that are homozygous for the mutations, along with six individuals that are homozygous for the wild alleles from the BC2F2 for minimizing the background effect.

Progress 12/01/17 to 11/30/18

Outputs
Target Audience:1. Scientific community at large; 2. Plant biologists andwheat scientists; 3. Wheat growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Drs. Lei Hua and Zhengzhi Zhang continued their work on this project in the labs of Dr. Li and Yang at South Dakota State University (SDSU) and Iowa State University (ISU), respectively. Lei Hua, previously trained in rice biology and soybean physiology, is working to develop and refine the mutation detection protocol, identify the mutations, and conduct wheat transformation. Zhengzhi Zhang, previously trained in genome mapping, is working to establish the CRISPR/Cas9 system and construct GS-specific CRISPR/Cas9 constructs for Agrobacterium-mediated wheat transformation, particularly the double Cas9 vectors and multiplex sgRNA cassettes. Graduate student Ajay Gupta joined Li Lab in spring of 2018. He is working on the identification of mutations. Drs. Lei Hua and Zhengzhi Zhang and Mr. Ajay Gupta 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 1 for the transformation of the newly developed constructs into FDR wheat, 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. Ajay Gupta will work on Objective 2 and Objective 3 to evaluate the phenotype effects of the mutations. How have the results been disseminated to communities of interest??Results have been disseminated to the research community through journal publications, scientific conferences, and other meetings as described in the products section. 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 the GS candidate genes 1. Check the potential off-target sites with our existing target site with Cas9 to address the reason why the Cas9 is not strong enough in some cases 2. Develop base editing constructs and test their efficiency. Objective 2. Identify mutations in the GS and GN candidate genes We will screen the T0 and T1 transgenic populations for beneficial mutations and transgenes as follows: 1. Continue to genotype T1 and T2 transgenic plants for edit mutations, particularly from the B-genome homoeolog, in TaGLW7 and TaGW2 mutations using our established procedure, (i.e., PCR-RE, PCR-Cas9 RNP assay, validation by Sanger sequencing). 2. Screen the T1 populations from the remaining T0 plants for TaDEP1, TaTGW6-3.1, TaTGW6-3.2, and TaTGW6-7.1. 3. Screen the T0 and T1 transgenic plants for new multiplex targeting constructs with two Cas9 transgenes for desired mutations. 4. Backcross the desired mutants of TaCKX2-1, TaGLW7, TaGW2 to wild type FDR for transgene null mutants. 5. Combine the mutations from homologous loci for triple mutations for GS and GN. We have established an efficient and accurate procedure for genotyping mutant alleles using the Cas9 RNP. We started to cross the plant carrying beneficial mutations of TaCKX2-1 with elite wheat cultivars, such as For Front and Prevail from South Dakota State University. We are in communication with the IWYP hub to transfer the beneficial mutant alleles into its elite lines. This will accelerate transferring the edit mutations into breeding populations. Objective 3. Characterize the effect of mutations on GS and GN candidate genes In the coming year, this part of the research will focus on the phenotypic characterization of TaCKX2-1, TaGLW7, and TaGW2 (Objective 3.1). The homozygous mutants will be measured along with wild type FDR for GS and GN, and statistical analyses will be used to examine the effect of the edit mutations on these traits. This research activity will help identify beneficial mutant alleles. We will also characterize the TaGLW7 mutations for their expression in spike and developing grain by quantitative RTPCR. Objective 4: Transfer beneficial mutations into elite durum wheat We will backcross the F1 hybrids of TaCKX2-1 mutants to Divide and select for the mutations and against the transgene and the D-genome chromosomes. We will cross and backcross the TaGLW7-A and TaGW2-A mutants and upcoming new mutants to Divide.

Impacts
What was accomplished under these goals? Objective 1: (80% accomplished) We generated 13 constructs in year 1, a majority of which have been transformed in wheat cultivar FDR. We also designed sgRNAs for 16 grain regulatory genes using a more rigorous algorithm to ensure that they only found a match in the target genes and include a restriction site close the cleavage site (3 bp upstream the PAM motif) for a faster and cost-effective mutant screening at a later stage. In Total, 30 sgRNAs were designed with two sgRNAs targeting one gene, except for GLW7 and GS3 for which only one sgRNA was able to be designed. These sgRNAs were used to develop four multiplex sgRNA cassettes, with each cassette containing eight sgRNAs targeting four genes. These four sgRNA cassettes have been mobilized into the Binary vector carrying the Cas9-coding gene and generated constructs pgWGS14, pgWGS15, pgWGS16, and pgWGS17. We have transformed the 13 constructs into Agrobacterium tumefaciens strain EH105 and submitted to UCDPTC for the transformation of FDR wheat. We have received 62 T0 transgenic plants. At the same time, we have established an in-house wheat transformation facility at South Dakota State University to conduct Agrobacterium-mediated transformation of FDR wheat. We have generated more than 45 T0 transgenic plants for pgWGS1 and pgWGS13. This will greatly help the project and reduce dependence on the service contract. Objective 2: (50% accomplished) We detected one mutation in TaCKX2-D1 by PCR and sequencing, which was due to a 1,160-bp deletion. No mutations were detected in TaCKX2-A1 and TaCKX2-B1. This could be due to low edit frequency in wheat. To test this hypothesis, we sequenced the PCR products by the HiSeq 2000 platform. A total of 1,101,752 raw reads were obtained from the deep sequencing, of which 486,890 reads met the quality standards. Both TaCKX2-A1 and TaCKX2-B1 showed a 5.2% mutation rate, while TaCKX2-D1 had a lower mutation frequency of 3.3%. Deletions occurred at much higher frequencies than insertions in all three homoeologous genes. Deletion frequencies were 4.0, 4.6, and 3.1% for TaCKX2-A1, TaCKX2-B1, and TaCKX2-D1, respectively. By contrast, insertion frequency at these loci were 1.2, 0.5, and 0.2%, respectively. There are also rare cases of complex editing (simultaneous deletion and insertion) in TaCKX2-B1 and TaCKX2-D1, but they all happened at less than 0.05%. The length of deletion ranged from 1 to 37 bp in TaCKX2-A1, 1 to 47 bp in TaCKX2-B1, and 1 to 72 bp in TaCKX2-D1. Interestingly, a 37-bp deletion from position 129 to position 165 occured at a high frequency. It is the most abundant mutant allele in TaCKX2-A1 and TaCKX2-B1, with 22.5 and 19.3% of the total mutations, respectively. This deletion happens at 10% in TaCKX2-D1, ranking the third most abundant mutation next to the 1-bp deletion at position 165 (16.7%) and 2-bp deletion at positions 154 and 155 (12.6%). In TaCKX2-A1 and TaCKX2-B1, the 1-bp and 2-bp deletions at 3 bp upstream of the PAM sites of two sgRNA rank the abundance just next to the 37-bp deletion. Overall, the deletion frequency at nucleotides close to TaU6.3 driven sgRNA2 PAM sites is higher than that at nucleotides close to sgRNA1 PAM. This suggests that TaU6.3 may function stronger in transcription of sgRNA or sgRNA2 in TaCKX2-1, providing better capability to direct mutations. These results showed that our CRISPR/Cas9 system did work in the T0 transgenic plants. We screened T1 progeny carrying the Cas9 transgene for targeted mutations by a 3-step protocol. First, gene-specific PCR products were separated by agarose gel electrophoresis to detect large deletions. Second, the PCR products were digested by restriction enzymes (RE), if an RE recognition site was located in the target sequence and close to the PAM motif. Otherwise, the PCR products were digested by T7E1. We recently developed a very effective protocol to use the Cas9/gRNA ribonucleoprotein (RNP) to screen mutants. With the in-house purified Cas9 protein and in vitro transcribed guide RNAs, we prepared Cas9/gRNA RNP for each specific target site and used the RNP to screen mutants by digesting the relevant PCR products. RNPs cleave the wild type derived PCR product into two fragments but do not cut the mutated allele derived PCR product due to mismatch of gRNA and mutant. Finally, we validated the mutations through Sanger sequencing. We screened 1,374 transgenic plants of 10 constructs and performed 4,052 PCR-RE, PCR-T7E1, or PCR-Cas9 RNP assays, and identified 64 mutations for four orthologous genes (TaCKX2-1, TaGLW7, TaGW2, and TaGW8) from 485 plants of T0, T1 and T2 generation with an estimated average edit rate of 13.2%. From 231 T2 plants from the #T1-6 family, we detected 48 new mutations by PCR-RE with an estimated edit rate of 20.8%. In this family, we detected two homozygous mutations in a single generation. Our results showed that deletions are the dominant type of edit mutations in wheat. In the T0 plants, deep sequencing indicated that over 99% of the mutations were due to deletions. Our genotyping data of the T1 and T2 plants showed that only four insertions were found among the 64 mutations detected, with the remaining 60 mutations being deletions. This greatly contrasts with what was found in the model grass rice, where insertion is the primary type of edit mutations. We also investigated the inheritance of induced mutations in a T2 generation. The segregation ratios fit the 1:2:1 expectation (P > 0.14370) for TaCKX2-A1, TaCKX2-B1, TaGW2-6A, and TaGW2-6D. In the T2 populations, segregations of mutant alleles from the CRISPR/Cas9 transgene was observed, and Cas9-free mutants for TaGLW7 and TaGW2 have been selected and are scheduled for release to breeders after seeds increase. As we have not yet transformed the new constructs for multiplex targeting, we focused on selection of homozygous mutations of individual homologous genes to combine them into double or triple mutations. We have crossed the TaCKX2-A1 mutants with TaCKX2-B1 and TaCKX-D1 mutants, and expect to obtain homozygous double and triple mutants in early spring of 2019. We also crossed mutants TaGW2-A with mutant TaGW2-D. Objective 3: (8% accomplished) While we are combining the mutations in the homologous copies of an orthologous gene or different orthologous loci, we evaluated the effect of mutation of the 1,160-bp deletion in TaCKX2-1D on grain number per spikelet and per spike, as well as spikelet density. Results showed that there was no significant difference in spike length, spikelet number/spike, or spikelet density between the mutant and wild type segregants from the same T2 population (P > 0.76805). Compared to the wild type, the mutation increased grain number/spike from 40 to 57 (P = 0.01856) and grain number/spikelet from 2.4 to 3.4 (P = 0.00331). This indicated that TaCKX2-1 negatively regulates the trait of grain number in wheat. This is consistent with study in rice, where reduction-of-function or loss-of-function mutations in OsCKX2 (Gn1) increased grain number per panicle. Objective 4: (5% accomplished) We will transfer the GS/GN mutants identified in FDR with the elite durum cultivar Divide and backcross the hybrids to Divide. 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 crossed FDR and Divide, and their F1 hybrids grew well and did not show the necrosis phenotype, indicating that the GS/GN mutations developed in FDR can be transferred into durum. We have crossed the mutants for TaCKX2-A1 and TaCKX2-B1 with Divide, and the F1 will be genotyped for the mutant alleles and CRISPR/Cas9 transgene and backcrossed to Divide in fall of 2018.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hua L, Zhang Z, Yang B, Li W. 2018. CRISPR-based genome editing of grain size regulators in wheat. ASPB MidWest Conference. 3/3/2018 to 3/4/2018. Ames, Iowa. P29
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Zhang Z, Hua L, Gupta A, Tricoli D, Edwards KJ, Yang B, Li W. 2018. Development of an Agrobacterium-delivered CRISPR/Cas9 system in wheat. Submitted to Plant Biotechnology Journal


Progress 12/01/16 to 11/30/17

Outputs
Target Audience:1) Geneticists, genome biologists and scientific community at large. 2) Wheat geneticists and breeders. 3) Wheat growers. Changes/Problems: Nothing Reported 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.

Impacts
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

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Li W, Yang B. 2017. Translational genomics of grain size regulation in wheat. Theoretical Applied Genetics. 130:17651771.