Overview

Our project aimed to increase the amylose content in sweet potatoes to produce a new anti-glycemic food/food ingredient. Sweet potatoes are polyploid plants, and in addition, a gene can have multiple homologous genes located on different chromosomes. We achieved multiplex gene knockout in sweet potatoes. Common important crops in plants, such as wheat, maize, and sugarcane, are also polyploid. Our method can provide reference ideas for future iGEM teams conducting research in plant synthetic biology through gene editing. Additionally, improving the hereditary traits of crops is a lengthy and ongoing process. Our engineered sweet potatoes with high amylose content provide better chassis characteristics for further modifications by future teams. We have also conducted research on current government policies and the market regarding anti-glycemic foods, which can serve as a reference for subsequent iGEM teams.


We have achieved the following contributions.

CRISPR-Cas9 Multi-Gene Knockout System for Sweet Potato

This multi-gene knockout system is designed for use in sweet potatoes and can serve as a valuable resource for future teams. The construction of the vector involves two key steps.

  1. First, the sense and antisense single-stranded sgRNAs are annealed to form double-stranded DNA, which is integrated into the linearized primary vector psgR-Cas9-At using T4 DNA ligase. This primary vector contains the cas9 gene, nuclear localization signals, and a detection tag.
  2. After verifying the assembly of the recombinant plasmid, the second step involves excising the sgRNA and cas9 expression cassettes with restriction enzymes, recovering them, and transferring them into the shuttle plasmid pCambia1300. This vector includes a hygromycin resistance gene for positive selection of transgenic plants.

For simultaneous knockout of multiple genes, sgRNAs targeting different genes are individually inserted into the primary vector psgR-Cas9-At, and then integrated into the same vector by restriction enzymes. Finally, this is transferred into the pCambia1300 vector, sharing a single cas9 expression system.

Key Considerations: Pay special attention to the efficiency of sgRNA design to ensure specificity and reduce off-target effects.

Descriptive Text
Descriptive Text
Descriptive Text

Figure 1. a. plasmid map of psgR-Cas9-IbSBEI-sgRNA; b. plasmid map of psgR-Cas9-IbSBEII-sgRNA; c. plasmid map of psgR-pAtU6-sgRNA SBEI-pAtUBQ-Cas9-tUBQ-pAtU6-sgRNA SBE II; d. plasmid map of pCAMBIA-1300--IbSBEI-sgRNA-Cas9-IbSBEII-sgRNA; e. Schematic diagram of the final vector pCAMBIA-1300--IbSBEI-sgRNA-Cas9-IbSBEII-sgRNA construction

Built high-amylose content strain of sweet potato as future chassis[SS1]

We have developed a sweet potato variety with high amylose content, which can serve as a chassis organism for future breeding improvements or for further development of anti-glycemic foods. Improving the hereditary traits of crops is a lengthy and ongoing process. Our engineered sweet potatoes with high amylose content provide better chassis characteristics for further modifications by future iGEM teams. For example, further improvements can be made to enhance its yield and taste through genetic engineering. Below is the characterization of this new strain:

Descriptive Text
Descriptive Text

Figure 2. a. Comparison of potted seedlings between genetically edited sweet potatoes and the WT; b. Comparison of internode length; c. Comparison of stem diameter; d. Analysis of SBEI and SBEII gene expression in genetically edited sweet potatoes.

Descriptive Text
Descriptive Text
Descriptive Text

Figure 3. Starch content analysis

Background Information, Policies, and Market Survey Summary on Anti-Glycemic Foods

The information we have gathered provides significant reference value for future iGEM teams. Firstly, diabetes is an increasingly severe global health issue, currently affecting over 537 million adults, with projections suggesting this number will rise to 643 million by 2030 (International Diabetes Federation). Thus, there is an urgent need to develop anti-glycemic foods.

Secondly, we have identified resistant starch as a key ingredient for anti-glycemic foods due to its beneficial effects on blood sugar control and gut health. High-amylose sweet potatoes, selected for their dietary fiber content and relatively favorable taste, show considerable application potential. Although high-amylose grains exist, their texture issues limit market applications, presenting avenues for future research improvements.

From a policy perspective, China is gradually relaxing regulations on genetically modified crops, opening new opportunities for gene-edited plants. For instance, the 2023 guidelines for the review of agricultural gene-edited plants indicate that such crops are gaining governmental approval.

Market analysis reveals that 88% of the public believes that diet significantly impacts health, and 92% are willing to pay a premium for anti-glycemic foods, indicating substantial market potential for these products.

This information will assist iGEM teams in making informed decisions regarding product design, market positioning, and policy navigation. Our investigation into the background, policies, and market dynamics of anti-glycemic foods offers vital insights for future iGEM projects, emphasizing the growing demand for such solutions considering the rising prevalence of diabetes.