Firstly, it should be noted that our experimental design is based on the optimized chassis - the wild-type tetraploid, and compared with the wild-type diploid to evaluate the level of each experimental design step. Because in addition to improving the tolerance of the chassis to genetic modification mentioned in the project background, we have also preliminarily judged that if regulatory targets are designed on wild-type diploids, the negative regulation of target traits by targets is ineffective because the increase in polyploidization is greater than that of gene editing and there is no selectivity. Specifically, we hope that the glutelin content will be basically unchanged and the globulin will be removed, but if we design the regulatory target on the wild type diploid, the predicted results will not be achieved. Because the target's negative regulation of glutelin and globulin content will be covered by multiple folds, resulting in content increase, which is inconsistent with the expectation of glutelin content and completely contrary to the expectation of globulin content. We have further evaluated the reliability of this judgment in the engineering process. (Please read project-engineering-2nd group model)

• The first step is experimental design

The core goal of the first step is to find the regulatory target of the expression group: to adjust the proportion of each subtype in each subfamily of glutelin, and to adjust the relative content of glutelin and gliadin, and to remove globulin; This design only focuses on the content of various proteins and lysine, and temporarily does not pay attention to the structure of proteins. Therefore, the design can be guided by a linear regression model.
This expression group regulates the target (modeling guidance requires two mutant target genes to achieve our expected effect, one main effect and one auxiliary) (please read the project engineering linear regression model for predicting and comparing the effects of different sequences of polyploidization and gene editing)

1. Screening for main effect mutation target genes

The main effect mutation needs to achieve the following functions:

1.1 Rice protein either increases hydrophilicity or becomes lipophilic (either promoting protein water absorption and starch gelatinization, or utilizing protein lipophilicity to promote fat emulsification).

That is to say, to reduce the expression of glutelin that is not fat soluble but poorly water-soluble, we need to select target genes in glutelin B or C or D families.

1.2 Increase in the total number of lysine residues in rice protein

Increase the lysine content of protein and align its essential amino acid ratio with the ideal amino acid pattern proposed by WHO (Trp: Lys: Leu: Phe: Ile: Thr: Met: Val=100:120:90:70:85:60:45:65)

The average content of lysine residues in the glutelin family is much higher than that in the gliadin family. It is necessary to increase the compensatory expression of lysine rich subtypes in as many glutelin subfamilies as possible (regardless of A, B, C, D subfamilies). And because the content of lysine residues in glutelin family is not low, but the original transcription level of OsGluB1 is the lowest in the main gene of glutelin transcriptional regulation network, the mutation can relieve the pressure of compensatory expression of other lysine rich glutelin in glutelin family, and ensure that they can compensate for the loss of lysine residue content of OsGluB1 which is not low but cannot be expressed.

Ziyan Shen.(2022, June 06).Moderate regulation of rice grain protein content by simultaneous editing of glutelin synthesis genes using the CRISPR／Cas9
system.

Therefore, OsGluB1 was chosen as the main effect mutation target gene for expression group regulation, and CRISPR/Cas9 gene edited target genes were used for targeted editing.

2. Evaluation of the regulatory effect of target expression group composed of main effect mutations

But when only one target gene is mutated, the predicted results of the dry experiment are no longer ideal. We had originally decided to find the reason to correct this step in the experimental design, but we still hesitated.
After all, this is the first mutated target gene we have chosen, and whether the effect of its mutation can reach the expected level will directly determine whether we need to choose the second mutated target gene to assist in forming the expression group regulatory targets of the two target gene mutations. If the wet experiment results are ideal, we do not need to mutate the second target gene, which is more conducive to achieving the expected results and protecting the physiological functions of rice from interference. Therefore, we still conducted a wet experiment, but the results were indeed unsatisfactory, which is why we gave up on this experimental design. (Please read Wet Lab result)
We analyzed the reasons for the poor regulatory results of the expression group with only major effect mutations based on the priority of meeting expected outcomes (nutritional quality>food safety>taste quality).
In terms of nutritional quality, if we only consider the extremely significant increase of glutelin content, it really meets the expectation, but the biggest limiting factor of rice protein nutritional quality is the lysine content, and the result of lysine content decline obviously goes against this point.
In terms of food safety, glutelin contains potential allergenicity, which also easily leads to intolerance. The extremely significant increase in its content is not in line with expectations. In terms of taste quality, the content of glutelin that is not conducive to improvement has not been further improved, and the content of gliadin that is conducive to improvement has not been further improved. Their relative content is further unbalanced, so it is also not in line with expectations.

3. Assisted mutation selection

We prioritize solving the problem of decreased lysine content based on the above analysis, and through further literature review, we have identified the cause.
The mutation of OsGluB1 resulted in the synergistic inhibition of the expression of two major genes OsGluA3 and OsGluB2 in the transcriptional regulation network of glutelin with high original expression levels. As a result, other lysine rich glutelin genes could not adequately compensate for the loss of lysine residue content of glutelin.

And the encoding single gene OsGlb of globulin, as an auxiliary mutation target gene, can precisely achieve two functions:

a. Remove the co inhibition of the expression of OsGluA3, the main gene of glutelin transcriptional regulatory network, which has higher expression than OsGluB1 and OsGluB2. And further enhance the expression level of the main genes OsGluA1 and OsGluA2 of the glutelin transcription regulation network and the lysine rich glutelin gene OsGluB7, so that the compensatory expression is sufficient to realize the compensation of the total residue content of glutelin lysine.

b. Knock out allergen globulin. Because there is not much residual globulin in rice endosperm after processing and globulin does not have interference from protein families, it is encoded by a single gene. As long as OsGlb is mutated, rice can almost not express globulin.

4. Evaluation of the regulatory effect of target expression group composed of auxiliary mutations

Fortunately, the experimental design of the expression group regulatory targets (OsGluB1 and OsGlb dual gene mutations) with the addition of auxiliary mutations was validated by dry and wet experiments, and was highly consistent with expectations.

• Step 2 Experimental Design: The core is to regulate the proteome

This design step involves the structure of proteins and requires guidance from protein structure prediction and molecular docking models.
We have identified OsGluB1 and OsGlb mutations as regulatory targets, which require targeted editing using CRISPR/Cas9 gene editing technology for implementation. The identification results of the mutations indicate that both have undergone frameshift mutations. The prediction results of protein primary structure indicate that the termination codon of OsGlb appears one reading box ahead of schedule, while the termination codon of OsGluB1 is severely delayed. (Please read the model model summary: Prediction Results of Primary Structures for Wild Type and Mutant Glutenin B1, and Globulin), which means that this double mutation regulatory target not only leads to changes in the ratio of rice storage protein components and glutenin subtypes and lysine content, but also leads to fundamental changes in the properties and functions of glutelin with little change in content and globulin with very little expression. In order to improve breeding efficiency, we used dry experiment results to evaluate whether this frameshift mutation is expected to be beneficial.
Nutritional value assessment, please read
Project-engineering-3d group model Changes in Nutritional Value
Please read the food safety assessment
Project-engineering-3d group model changes in food safety
Project-engineering-4th group of models
Please read for taste quality assessment
Project-engineering-3d group model Taste changes
Project-engineering-3d group model - Bird level structure prediction

• Future Work Design

Biosafety protection system (please read project safety product safety)

Reference

[1] Ziyan Shen I. Moderate regulation of rice grain protein content by simultaneous editing of glutelin synthesis genes using the CRISPR/Cas9 system[D]. Yangzhou: Yangzhou University, 2022.
[2] Xiaomei Wang. Research of Orientation Mutation of Glutelin Gene GluA3 in Rice (Oryza sativa L.) by CRISPR/Cas9 System[D]. Shanghai: Shanghai Normal University, 2021.
[3] Niu Hongbin. Cloning, Expression and Characterization of Genes Related to Glutelin in Rice[D]. Jiangsu: Nanjing Agricultural University, 2005.
[4] Fangyuan Chen. Screening and functional studies of genes related to seed storage protein synthesis in rice[D]. Shandong: Normal Shandong University, 2023.
[5] Cai Detian, Chen Jianguo, Chen Dongling, et al. Breeding of two polyploid rice lines with polyploid meiotic stability [J]. Chinese Science Series C, 2007, 37(2):217-226. DOI:10.3969/j.issn.1674-7232.2007.02.013.
[6] YANG Xue-zhi, HAN Yun-zhe, PIAO Xue-mei, XU Wei-hao, BAI Xue-feng, LIU Hong-liang*. (Yanbian Korean Autonomous Prefecture Academy of Agricultural Sciences, Yanbian Jilin 133400, China DOI:10.3969/j.issn.1673-6737.2024.03.012.
[7] Yihua Wang. Map-based Isolation and Characterization of Key Genes Involved in the Glutelin Biosynthesis Pathway in Rice[D]. Jiangsu: Nanjing Agricultural University, 2007. DOI:10.7666/d.Y1814799.
[8] Xian Lin. Determination of Endosperm Protein Content during Different Development Stages and Gluten Spectrum Analysis in Different Ploidy Rice (Oryza sativa L.). [D]. Guangzhou: South China Agricultural University, 2018.
[9] Rice-Based Gluten-Free Foods and Technologies: A Review - PubMed (nih.gov)