Progress 01/15/21 to 01/08/24
Outputs
Target Audience:The target audience includes the scientific community at large, plant biologists, wheat scientists, and wheat growers. Changes/Problems:As mentioned above, we had difficulties installing the MRE mutation to GRF4 via the prime editing in Objective 1, then adopted the TR-HDR approach, and succeeded. What opportunities for training and professional development has the project provided?This project trained three research scientists, a graduate student and a technician in genome editing, wheat genetics, and molecular biology. Research assistant Ajay Gupta worked on this project until end of July 2021 in the development and test of the PE systems, GRF4 PE constructs, wheat transformation, and construction of the deep sequencing libraries. Research assistant Yifan Wang worked on screening the TR-HRD populations for MRE mutations. Postdoc scientist Dr. Ming Ma worked on nitrogen use efficiency of the GRF4 mutant via in-kind support. MS graduate student Ankush Sharma has been working on the project since August 2021 in analysis of the deep sequencing results and screening the T1 populations for mutations. Technician Yanhang Zhang worked on wheat transformation. How have the results been disseminated to communities of interest?Results have been disseminated to the research community mainly through scientific conferences/meetings, and the project webpage as described in the products section. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Create synonymous mutations in the MRE to over-express GRF4. (100% Accomplished) 1. Develop prime editing systems. In this project, we have developed four prime editing (PE) systems. The first two PE systems were built by fusing Cas9 (H840A) nickase with the M-MLV reverse transcriptase (RT) or with cauliflower mosaic virus (CaMV) RT. Six constructs were developed by integration three pegRNA/RT-PBS cassettes to target GRF MREs from chromosomes 2A/6B and 2B/2D, which are discriminated by an SNP. The pegRNA cassette is driven by a strong, composite promoter 35S-CmCYLV-U6. These PE systems worked in protoplasm-based reporter gene-targeting experiments, but only low-level somatic mutations were detected in 29 T0 transgenic plants by deep sequencing. To increase the efficiency of prime editing, we developed two more PE systems, i.e., the third and fourth systems. The third system involves use of the engineered pegRNAs (epegRNAs), in which the MLV pseudoknot sequence was added to the pegRNA/RT-PBS cassette at its 3' end and integrated with pH-ePP vector, which worked in rice and maize, and later in wheat. The fourth system is the PEmax, an optimized PE2 protein harboring an nCas9 variant of increased nuclease activity, an additional NLS, and a new linker between nCas9 and RT domains. PE3max, a PEmax derivative using a pegRNA with evopreQ1 appended to its 3' end (pegRNA-evopreQ1). We developed 15 T0 transgenic plants for the third system and over 100 T0 transgenic plants for the fourth system. Screening the T0 and T1 populations of the first system did not detect mutation. We detected mutations in the T0 plants of the second system by PCR using a primer targeting the mutation, but Sanger sequencing of the PCR products of the target interval cannot confirm the mutation, suggesting that the PCR signal detected was due to low frequency of somatic mutation. Although both systems worked for rice and the first worked for wheat in recent publications, they showed very low efficiency for editing the miR396 MRE if it works. This is due to the genetic redundancy of the MREs in the wheat genome and no other targets to be selected. As a result, the sgRNA found perfect matches in nine GRF homoeologous genes and six off targets with a 1-bp mismatch at the 3' end. This may have diluted PE efficiency at each target site. 2. Precision editing MREs of GRF genes by CRISPR-mediated TR-HDR. After frustration with prime editing the MREs, we established a CRISPR-mediated tandem repeat (TR)-homology-directed repair (HDR) system to install the MRE mutation to the GRF genes. The TR-HDR system was initially developed in rice. In the system, a 5?-phosphorylated double-stranded oligodeoxynucleotide (dsODN) is used as donor DNA carrying the desired edits, which is first inserted at the designated genomic target via non-homologous end joining and creates a tandem repeat and reconstitutes the target site upstream. CRISPR/Cas9 subsequently generates a break at the reconstituted target site between the two repeat units, triggering high-efficiency HDR to replace the original sequence with the donor DNA (Nat Biotech 38: 1402-1407). The nine GRF genes containing the perfect target are GRF4 on the group-2 chromosomes, GRF5 on the group-7 chromosomes, and a GRF orthologous gene on the group-6 chromosomes, which are negatively regulated by miR396. Approximately 3,000 immature embryos of wheat cultivar Fielder were bombarded with gold particles coated with the CRIPSR construct carrying Ubi-Cas9 and sgRNA targeting the MRE and donor DNA, 104-bp 5?-dsODN, which is identical to the GRF genic sequence downstream of the cleavage site and containing synonymous mutations at nine positions near the cleavage site. Screening ~300 T0 plants by PCR found 12 of them carrying the insertion of the donor DNA. Genotyping by PCR and Sanger sequencing of the 8 T1 populations has identified synonymous MRE mutations on GRF4, GRF5, and the GRF gene on group-6 chromosomes. 3. Effect of the mutation on GRF4 expression. We use a dual luciferase assay to quantify the repression of GRF4 by microRNA396 (miR396), in which a wheat miR396 gene is over-expressed, and the wild-type and mutated forms of the MRE or its mutation are tagged to the downstream of a firefly luciferase reporter. Compared to the wild type, the synonymous mutations MRE mutation increased the target sequence by 4-fold, indicating that the miR396 binds to MRE in wheat, and disruption of the MRE led to GRF4 overexpression. 4. Impact. The development of the PE systems adds a new tool to wheat genome editing although they did not work well for the high-copy targets like miR396 MRE. Establishment of the TR-HDR provides a choice for precision editing of wheat genes, particularly for the targets of high copy number. Development of the MRE mutations in the GRF genes will provide an opportunity for functional characterization. Objective 2. Evaluate the mutation effect on yield components and NUE (100% Accomplished) 1. Phenotyping the GRF4 MRE mutation. The mutant plants were crossed and backcrossed to the wild-type (WT) Fielder to purge the transgene. The F2 or BC1F2 populations were genotyped for the mutation and Cas9 transgene, and the homozygous mutants were selected and phenotyped together with the WT. The seeds of Fielder and the mutant were germinated in water. The seedlings were transplanted in 3" square pots and 6" round pots supplied with 1.6 g and 8.3 g of Multicote 4 controlled release fertilizer, respectively. The pots were placed in a greenhouse room without spacer. While there is no difference in plant height for plants in the small pots, the plant height was slightly reduced for the mutant when grown in the large pots (p = 0.00615). The mutant plants flowered 2.6 days earlier in big pots and 4.0 days earlier in small pots as compared to the WT (p < 0.00391). The mutation increased 0.5 spikes per plant when grown in small pots (dense condition) (p = 0.00306). When grown in large pots, no difference was detected for spike/plant and spikelets/spike (p > 0.48901), but grains/spike and grains/spikelet were increased by 11% in the mutant as compared to the WT (p < 0.00146). In N15-feeding experiments, the GRF4-2B mutant showed an increase of N uptake in shoot by 1.9-fold at 30 min (p = 0.00164) and 2.5-fold at 60 min (p = 0.00012) as compared to the WT, indicating that MRE mutation increased NUE. 2. Impact. The identification of the MRE mutation effect on flowering date, spike number per plant, grain number per spike (spikelet), and NUE laid a knowledge foundation for improving wheat agronomic, yield, and NUE traits by engineering the GRF4-mediated network. Objective 3. Develop breeding-ready germplasm (100% Accomplished) 1. Transfer MRE mutation into elite wheat cultivar. We have crossed and backcrossed the GRF4-2B MRE mutant plants with SD spring wheat Boost and Prevail. The BC1 plants were screened using mutant-specific primer, and the positive plants were selected for further backcrossing and the Cas9 transgene was selected against. Sanger sequencing showed that the GRF4-2B MRE mutation transmitted in Boost and Prevail background. The transgene-free MRE mutant lines are ready to be used for wheat breeding. 2. Impact. The transgene-free MRE mutant lines in elite wheat background together with the mutation-specific marker are readily used for wheat improvement as novel germplasm.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Li W. 2024. Precision editing of Growth Regulation Factor genes by CRISPR-mediated HDR in wheat. International Plant and Animal Genome Conference (PAG 31), Jan 12-16, 2024. San
Diego, CA. PO0391.
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The target audience includes the scientific community at large, plant biologists, wheat scientists, and wheat growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained two graduate students in genome editing, wheat genetics, and molecular biology. MS graduate student Ankush Sharma has been working on the project until May 2022 in the analysis of the deep sequencing results and screening of the T1 populations for mutations. Ph.D. student Ahmed Alhusays worked on the project but was supported by other funds as an in-kind contribution, screening the T0 and T1 populations for mutations. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Dr. Yifan Wang is expected to join the project in late January 2023 and work as a postdoc scientist to strengthen the research activity. The plan for the coming year will be as follows. Objective 1. Create synonymous mutations in the MRE to over-express GRF4. We will continue to screen all the T1 populations derived from the epegRNA constructs for mutations by PCR assay and Sanger sequencing. We will continue to transform PE3max constructs into Fielder and screen the mutation by PCR-restriction assay and Sanger sequencing. We will continue to improve the PE systems based on the advancements in human and rice studies. Objective 2. Evaluate the mutation effect on yield components and NUE. We will phenotype homozygous mutants for yield components and nitrogen assimilation efficiency. Objective 3. Develop breeding-ready germplasm. We will backcross the F1 hybrids between inheritable GRF4 mutants and wheat cultivar Prevail to eliminate the transgene based on marker genotypes.
Impacts
What was accomplished under these goals?
Objective 1: Create synonymous mutations in the MRE to over-express GRF4. (40% Accomplished) 1. Develop prime editing systems. Previously, we developed two prime editing (PE) systems in which the Cas9 (H840A) nickase was fused with the M-MLV reverse transcriptase (RT) or with cauliflower mosaic virus RT. We also developed a pegRNA cassette, in which a single pegRNA/RT template-primer binding site (RT-PBS) pair can be cloned and flanked by tRNA-Gly and HDV ribozyme sequences to enhance the cleavage of the pegRNA/RT-PBS and transfer to the meristem. The pegRNA cassette is driven by a composite promoter 35S-CmCYLV-U6. We detected a mutation by deep sequencing and Sanger, but it was not able transmitted to the next generation, indicating the possibility of somatic mutation. To increase the efficiency of prime editing, we developed two more systems based on the advancement in humans from Dvid Liu's group and in rice, the model cereal, while continue to screen the T1 and T2 populations from the previous year. One of the newly developed systems involves use of the engineered pegRNAs (epegRNAs), in which the MLV pseudoknot sequence was added to the pegRNA/RT-PBS cassette at its 3' end. The study in human cells showed that the pseudoknot can protect the pegRNA/RT-PBS from degradation by the 3' exonucleases and increase the efficiency of prime editing by approximately three-fold. The second system is the PEmax, an optimized PE2 protein harboring an nCas9 variant of increased nuclease activity, an additional NLS, and a new linker between nCas9 and RT domains. PE3max, a PEmax derivative using a pegRNA with evopreQ1 appended to its 3' end (pegRNA-evopreQ1), worked well in rice. Thus, we adopted it for wheat. We also replaced 35S-CmCYLV-U6 with the 35S-CmCYLV-U3 promoter to drive the pegRNA/RT-PBS cassette. 2. Develop GRF4 PE constructs. With the new systems, we developed four pegRNA/RT-PBS cassettes, two with MLV pseudoknot and two with evopreQ1 appended to its 3' end. These pegRNA/RT-PBS cassettes contain an RT template to introduce synonymous mutations in nine positions of the 20-nt miR396 recognition element (MRE) of the GRF4 homoeologs on chromosomes 2A/6B and 2B/2D due to an SNP that discriminates the GRF4 targets. The resultant epegRNA cassettes were mobilized into the H840A-MLVRT binary vector, and the pegRNA-evopreQ1 cassette were mobilized into the binary vector containing PE3max by Gateway recombination. 3. Wheat transformations. All the four constructs have been transformed into wheat cultivar Fielder by Agrobacterium mediation. The transformation of the epegRNA constructs generated 21 T0 transgenic plants and three T1 populations, while the transformation of the PE3max constructs is at callus-induction stage. 4. Screening PE mutations. Previously, we identified a mutation at the +1 position in the T1 populations. Screening of 400 plants of four T2 populations failed to recover it, suggesting that it was a somatic mutation in the T1 plants. For the newly developed transgenic plants of the epegRNA constructs, we screened the T0 and T1 populations using a primer specifically targeting the designed mutation nucleotides in combination with the homoeologs-specific primers. The PCR amplified a GRF4-2D fragment of expected size in two of the T0 plants, but did not amplify in the non-transgenic Fielder as a control. Of the first batch of 27 T1 plants screened, the PCR identified three T1 plants positive for GRF4-2A, two for GRF4-2B, and two for GRF4-2D. Sanger-sequencing of PCR products containing the target site from the positive plants did not detect the mutation. This would suggest that the mutation rate is much lower in wheat compared to rice, and PCR amplification may arise from somatic mutation in a small number of cells, which can be detected by deep sequencing but not by Sanger sequencing. 5. Effect of the mutation on GRF4 expression. We use a dual luciferase assay to quantify the repression of GRF4 by microRNA396 (miR396), in which a wheat miR396 gene is over-expressed, and the wild-type and mutated forms of the MRE or its mutation is tagged to the downstream of a firefly luciferase reporter. Compared to the wild type, the PE mutation increased the target sequence by 5-fold, indicating that the miR396 binds to MRE in wheat, and disruption of the MRE led to GRF4 overexpression. 6. Impact. Compared to rice, wheat prime editing is much more challenging. The development of the new PE systems, constructs, and transgenic plants laid a foundation for wheat PE breakthrough. The upregulation of GRF4 by the MRE mutation suggests that the GRF4-miR396 module can be explored as a toolbox to improve wheat yield and NUE. Objective 2. Evaluate the mutation effect on yield components and NUE (0% Accomplished) No activities were conducted towards this objective. Objective 3. Develop breeding-ready germplasm (0% Accomplished) We have crossed the mutant plants with SD spring wheat Prevail but the mutation was not recovered in the F1. Thus, this objective was inactive until a new, inheritable mutation is identified.
Publications
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The target audience includes the scientific community at large, plant biologists, wheat scientists, and wheat growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained a research scientist and a graduate student in genome editing, wheat genetics, and molecular biology. Research assistant Ajay Gupta worked on this project until end of July 2021 in the development and test of the PE systems, GRF4 PE constructs, wheat transformation, and construction of the deep sequencing libraries. MS graduate student Ankush Sharma has been working on the project since August 2021 in analysis of the deep sequencing results and screening the T1 populations for mutations. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Create synonymous mutations in the MRE to over-express GRF4. We will screen all the T1 populations for PE mutations by PCR assays and the mutations will be validated by Sanger sequencing. Then we will measure the transcription of the mutant genes by qRT-PCR. Objective 2. Evaluate the mutation effect on yield components and NUE We will phenotype homozygous mutants for yield components and nitrogen assimilation efficiency Objective 3. Develop breeding-ready germplasm We will cross and backcross the GRF4 mutants, once identified, to spring wheat cultivars Prevail to eliminate the transgene.
Impacts
What was accomplished under these goals?
Objective 1: Create synonymous mutations in the MRE to over-express GRF4. (40% Accomplished) 1. Develop prime editing systems. We developed two prime editing (PE) systems, in which the Cas9 (H840A) nickase was fused with the M-MLV reverse transcriptase (RT) or with cauliflower mosaic virus (CaMV) RT, and optimized the codon usage for wheat. The wheat codon-optimized H840A-RT fusion genes are driven by maize ubiquitin promoter (Ubi) in a binary vector. We developed a pegRNA cassette, in which a single pegRNA/RT template-primer binding site (RT-PBS) pair can be cloned and the pegRNA/RT-PBS is flanked by tRNA-Gly and HDV ribozyme sequences to enhance the cleavage of the pegRNA/RT-PBS and transfer to the meristem. The pegRNA cassette was mobilized to the binary vector containing the H840A-RT fusion genes by the Gateway-mediated recombination and generated two PE vectors. The pegRNA cassette is driven by a strong, composite promoter 35S-CmCYLV-U6. To test the efficacy of this PE3 system, we built a reporter gene-targeting system for transient assay in wheat protoplasts, in which a nonsense mutation was introduced in the 5' part of the mGFP5 gene to create a premature stop codon, and the resultant dead mGFP5 (dmGFP5) gene is driven by the 35S promoter. The pegGFP and the H840A-RT fusion gene were integrated into one plasmid by Gateway recombination to generate a pPE-pegGFP plasmid. When the pdmGFP and pPE-pegGFP constructs are co-transfected into wheat protoplasts, the pegGFP/H840A nicks the antisense strand upstream the mutation site, and the CaMVRT corrects the mutation using the RT template. This restores the GFP function and lights up the protoplasts with green fluorescence, indicating that the PE systems are feasible for precise gene editing in the wheat cells. Subsequently, we also developed a peg cassette to clone two pegRNA/RT-PBS pairs. 2. Develop six GRF4 PE constructs. An SNP discriminates the GRF4 targets from chromosomes 2A/6B and 2B/2D. Two sgRNA genes were designed and paired with an RT-PBS, which writes synonymous mutation at seven positions in addition to position 1 from the cleavage site. These pegRNA/RT-PBS cassettes were integrated into the vectors containing H840A-MLVRRT and generated four constructs, two of which were for GFR4-2A/6B and the other two for GRF4-2B/2D. Later, we developed a double- pegRNA/RT-PBS cassette, which can host two pegRNA/RT-PBS pairs. Considering that the GRF4-2B/2D are expressed at a relatively higher level, a double- pegRNA/RT-PBS cassette was first used to target chromosomes 2B and 2D with the same pegRNA/RT-PBS. Our hypothesis is that the two copies of pegRNA/RT-PBS will increase its cellular concentration and eventually the PE efficiency. 3. Wheat transformations. All the six constructs described above have been transformed into wheat cultivar Fielder by Agrobacterium mediation. The transformation of the first set of the four constructs generated 29 transgenic plants, while the transformation of the second set of the two constructs produced 30 transgenic plants. 3. Deep sequencing analysis of the T0 plants. We screened the 29 T0 transgenic plants for PE mutations using the RT-PBS as a reverse primer and found that none of them carried the PE mutation. To gather insight into the somatic mutation types and frequency, we amplified the target region from chromosomes 2A, 2B, 2D, and 6B, and deep-sequenced the PCR products using the Hi-Seq technology. Deep sequencing generated 134,310 reads, of which 105,392 mapped to the target. Analysis of the105,392 mapped reads detected target modification in 3,230 (3.06%) reads, of which 2,564 (2.41%) reads had substitution, 640 (0.61%) reads had deletion, and 42 (0.04%) reads had both substitution and deletion. Almost all the substitution mutations happened at position 1. This result indicates the PE systems work in-planta and differs from the animal system, where PE can install longer mutations. 4. Screening PE mutations. Based on the deep sequencing results, we designed a new reverse primer with the 3' end targeting on the position-1 mutation. At the same time, we synthesize a DNA fragment containing the position-1 mutation as a positive control because no wheat cultivars carry the mutation. Screening 132 plants of four T1 families with either M-MLVRT or CaMVRT transgenes identified four mutations at position 1, two on GRF4-2B and two on GRF4-2D, with the frequency from 1 to 8%. Based on the sequencing chromatographs, the GRF-D4 mutations are homozygous and GRF-B4 mutations are heterozygous. 5. Impact. The development of the PE systems adds a new tool to wheat genome editing. The development of six constructs and 59 transgenic plants and the identification of the GRF4-2B/2D mutations laid a foundation for improving wheat NUE and grain yield by modulating the GRF4-mediuated pathway. Objective 2. Evaluate the mutation effect on yield components and NUE (0% Accomplished) No activities were conducted towards this objective in year 1. Objective 3. Develop breeding-ready germplasm (10% Accomplished) We have crossed the mutant plants with SD spring wheat Prevail.
Publications
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