1. Overview

With the continuous development of society, the existing natural biological fermentation productivity is not enough to meet the market demand. Therefore, genetic engineering has become increasingly important, and gene editing has gradually become one of the most important components of synthetic biology. In recent years, CRISPR has been the most popular technology for gene editing.

CRISPR was discovered in the early 1990s and firstly used in the biochemical experiments seven years later. As the third-generation gene editing tool, CRISPR/Cas system is based on simple RNA to guide DNA recognition. Due to its flexibility, convenience, low cost and high specificity, CRISPR/Cas has rapidly become the most popular gene editing tool. It has become a new favorite of gene editing and shown good application prospects in many fields such as genome engineering, genetic disease treatment, high-throughput functional genome screening, genetic disease diagnosis, etc. Therefore, in 2020, the Nobel Prize in Chemistry was awarded to Professor Emmanuelle Charpentier of France and Professor Jennifer A. Doudna for the development of a method for genome editing.

However, there are still some problems with CRISPR/Cas system such as off-target cleaves, which leads to CRISPR-based genome engineering strategy with variable editing efficiency for the different sites. This problem will affect the efficiency of high-throughput screening of the CRISPR edited cells. Therefore, we developed a CRISPR-based purification system “strainer”, which is an efficient and high-throughput CRISPR-based genome editing method to reduce the number of the unedited strains during the high-throughput genome editing, to further improve the efficiency of high-throughput genome editing, thus improving the construction efficiency of Escherichia coli (E. coli) strains. We utilized the double stranded DNA breaks (DSBs) as a signal to start the transcription of gRNA targeting on the plasmid harboring sacB gene. Then, only the strains with DSBs and recombineering could survive in the media with sucrose, because levansucrase encoded by the sacB gene converts sucrose to levan that is toxic to E. coli, thereby improving the overall editing efficiency during the genome engineering process.

We then used “strainer” for an isopropanol high-producing strain construction efficiently. “Strainer” can be potentially used as a universal method for cell factory construction.

2. Products and Customers

In recent years, the research on CRISPR has become very popular, and this method has been widely used. However, these methods generally have the off-target problems and cost a lot of man power and materials. Therefore, we want to change this situation by designing a tool, "strainer".

Based on CRISPR system, we used the dual-gRNAs and the toxic gene on three plasmids, combined with the SOS promoters, to remove the unedited primitive cells, which can improve the subsequent high-throughput screening efficiency and reduce the impact of the unedited cells on gene library construction, strain screening and data analysis.

Our target users include two parts:

The first part is the scientific research teams and the iGEM teams that need to use gene editing methods. The problem they are facing is that there are too many scrapped strains after high-throughput CRISPR-based gene editing, which leads to low efficiency of the subsequent screening, and expands need for the strains. The existing high-throughput screening methods are cumbersome, time-consuming, and costly. In addition, other CRISPR technologies generally have the off-target problems, which will greatly increase the screening cost. Therefore, it is difficult for customers to obtain a large number of plasmids by themselves, and it will also cost high to hand them over to other companies for production. So, we can use “strainer” to provide them with more efficient and much cheaper edited plasmids.

The second is the biological fermentation factories. Take the isopropanol fermentation factories as the examples. At present, isopropanol is mainly produced by chemical methods like acetone hydrogenation. There are also a few isopropanol biological fermentation factories. The output of the chemical methods is higher, but a large amount of waste water and waste gas need to be discharged, thus polluting the environment. In order to protect the environment and reduce carbon emissions, less-polluting biological fermentation methods appeared. However, the biggest disadvantage of the biological fermentation method is low output. To change this situation, we can modify the strains through the gene editing methods. For such customers, we can directly provide them with the edited E. coli strains according to their gene design.

We can use the "strainer" system for more efficient CRISPR-based gene editing. According to the plasmids required by the customers, the edited genes and donor DNAs, we can design the corresponding “strainer” ramification and cultivate them to obtain a large number of wild bacteria. We can use the CRISPR/Cas system for the high-throughput editing, and then obtain the target plasmid through gene sequencing and purification.

Compared with the general gene editing methods, our purification system has a lot of advantages in many aspects. First of all, CRISPR is simple, inexpensive and less cytotoxic than the other editing tools. Therefore, our project has great potential. Second, the requirements of the experimental environment are very easy to reach, and the requirements of the suitable environment for E. coli are not high. The screened strains can be obtained just in a short time under the normal environment. Third, the strains edited by "strainer" have undergone preliminary screening, leaving only the edited strains. The common gene editing methods will lead to the normal growth of strains which are off-target or even not edited, bringing a lot of trouble to the subsequent screening. Thus, we used the toxic proteins. We wanted the strains that have been successfully edited to grow normally, but the unedited strains to die directly. Therefore, we made the genes of the toxic proteins be recognized and cut by the Cas protein in the process of the gene editing, to ensure the edited strains can survive. In order to guarantee the accuracy of the identification, and considering the efficiency of the editing and latter production, we designed a three-plasmid system. Our "strainer" contains three plasmids. The first plasmid contains the lambda red system and Cas protein, which is used for DNA recombineering. The second plasmid contains the sacB gene, its product converts sucrose to levan, which is toxic to E. coli. The third plasmid contains two gRNAs. The gRNA-target is used for editing the target gene. The gRNA-toxic is induced by the SOS system for curing the plasmid harboring the sacB gene. When DSBs occurred, the SOS promoter of the second plasmid began to transcribe gRNA which targeted the sacB gene. The Cas protein guided by gRNA recognized and cut the sacB gene. Thus, the strains with successful recombineering can survive in the media with sucrose. The strains without DSBs, still has the plasmid harboring sacB gene, cannot survive in the media with the sucrose. This is why we can increase the overall editing efficiency. Fourth, we can reduce the cost of the subsequent screening. The cost of the gene sequencing is not cheap. We have reduced the number of the unedited strains, thereby reduced the workload of the subsequent gene sequencing and reduced the cost. Fifth, we can reduce the use of the number of the cells. Due to the limit of the current gene editing tools, the efficiency of the gene editing cannot provide enough successfully edited strains. Therefore, through our “strainer”, we can appropriately reduce the number of strains we need in the future.

Now, the technology is developed by the undergraduate team participating in the iGEM competition, which is hosted by the iGEM Foundation. We have completed the overall design of the project and established a feasible theoretical basis through literature research, quotation and verification. At the same time, we also carried out the basic verification experiments to prove the feasibility of the key steps in the process.

In the future, our tools and technique are likely to be more widely used and has high scope for development and improvement. At present, we have screened and removed the unedited strains. In the future, we can further study the gene insertion of the large fragments, the defects of CRISPR system delivery mode and other aspects.

3. Entrepreneurship Team

3.1 Company Profile

The business scope of the enterprise includes plasmid and strain production, technology research and development, technology transfer, technology consulting and technical services. In terms of organizational structure, the company adopts a flat model to avoid many disadvantages of hierarchical management, such as overlapping organizational levels, redundancy of personnel, and low efficiency of organizational operation, which speeds up the flow of information and is conducive to the refinement of management levels. The company consists of six departments: Research and Development(R&D), Marketing Department, Human Resources Department, Finance Department, Operation Department and Biosafety Department.

3.2 Company leaders and responsibilities

Executive director

Name: Wei Xiaomeng

Responsibilities: The executive director is responsible for the General Meeting of Shareholders, convening and organizing meetings of the board of directors, approving the work plan and work summary of the general manager, and formulating a reward and punishment system for the general manager. The chairman of the board of directors has the right to decide on the major issues of the company, such as investment, capital increase and reduction, etc.

Head of R&D Department

Name: Sun Yu

Responsibilities: Be responsible for the development of new products, the maintenance of relevant platforms, and the protection of intellectual property and technology. Be responsible for the design management, process management, scientific research and development planning. Prepare for the company's medium and long-term technological development plan and scientific research plan.

Head of Marketing Department

Name: He Qingwen

Responsibilities: Understand the industry situation and basic customer information, conduct market research and analysis, formulate the marketing plans according to the market situation and national policies, and promote sales.

3.3 R&D management

Based on R&D architecture design and various management theories, with the help of information platform, team building, process design and specific coordination of the execute management, cost management, project management, risk management and other activities, we comprehensively measure the quality, time and cost of R&D management. After the successful launch of new products, the company will carry out second-generation R&D according to product effects and market demand, and focus on R&D and market changes in stages according to the long-term development strategy.

3.4 Performance evaluation of department personnel

1. The assessment factors of management personnel are divided into: mutual evaluation within the department, project manager scoring and self-evaluation.

2. Proportion and calculation method of assessment factors:

Mutual evaluation within the department 50%

Project leader’s score 30%

Self-assessment 20%

3.5 Rewards and punishments

①　If the score is more than 90, a bonus of 15% of the salary will be paid.

②　If the score is lower than 60 for two consecutive months, 10% of the salary will be deducted.

4. Market and Competition

4.1 Market policy

The "Fourteenth Five Year Plan" for Bioeconomy Development issued on May 10, 2022 is China's first five-year plan for bioeconomy. It has made it clear that we should focus on making the bioeconomy bigger and stronger, and by 2025, the bioeconomy will become a strong driving force for high-quality development. Synthetic biology accounts for a large proportion in the biological field. Our project has promoted the development of synthetic biology, which is consistent with China's current policies.

4.2 Market scale and market structure

According to the report of Research and Markets, the compound annual growth rate of gene editing market has reached 31.1% from 2015 to 2022. The North American market has occupied the largest market of gene editing technology, followed by Asian, and European has been the third largest application market. Markets and Markets market research company estimated that the global gene editing market would grow from 2.84 billion dollars in 2016 to 5.54 billion dollars in 2019, with a compound annual growth rate of 14.3%. The global gene editing market would reach 8.1 billion dollars by 2050, of which CRISPR technology will become the gene editing technology with the fastest development and the highest share. Therefore, our system will have a good prospect.

4.3 Analysis of competitors and alternatives

At present, researchers in relevant professional fields are very enthusiastic about CRISPR high-throughput screening. In recent years, there have been many related patents, but each of these methods has their own shortcomings. At present, there are many companies at home and abroad that provide customers with the services of synthesizing plasmids and constructing bacteria, such as Kingsley Biotech, BioSci, etc.

Although the market competition is fierce, our technology is still very competitive in today's market due to its high efficiency and low cost.

5. Marketing and Sales

5.1 Product strategy

This product will first be piloted in some biological fermentation plants and other iGEM teams. After the success of the pilot unit, it will expand the popularity of the pilot unit through the Internet, and gradually increase the application of other strains in the process of expansion. We will further consolidate the system, improve user stickiness as well as market position and occupy market share.

5.2 Price Strategy

According to the market research conducted by the project team in the early stage, and considering the consumption capacity as well as willingness of the target customer group, the project was purposely designed to give full play to the long-term use advantages of the product, so as to develop products with short development time and low price, providing high-quality products.

During the trial period, the product will be launched at a preferential price of 80%.

5.3 Channel Strategy

Our promotion strategies are mainly divided into online and offline promotion.

5.3.1 Online channel

a. WeChat official account

The official account will popularize the knowledge of biosynthesis, gene editing and biological high-throughput screening, introduce the advantages of biosynthesis, help people understand the principle of genome engineering technology, eliminate the masses' doubts about modern biotechnology such as transgenic technology, reduce the distrust of biological products, strengthen the company's mass base, and better promote our products.

b. Long-term advertising

The project takes "market promotion” for our new product to enter the market as the primary task. We will use part of our proceeds to promote the product by live streaming and other online ways. For example, in QQ group chats, WeChat moments, we will cooperate with the third-party shop APP or the `related well-known websites to expand customer base and enhance brand awareness in a short time.

5.3.2 Offline channel

a. Campaign publicity

Hold some offline publicity activities, such as organizing or sponsoring biological competitions, organizing technical exchanges, and making charitable donations.

b. Promotion within professional units

At this stage, the project mainly serves the research institutions and the production plants. The platform will give full play to the publicity effects of these professional units at the social level, such as popularity, credibility, interpersonal relationships, etc. And work with them to promote marketing, so as to better integrate with the public. This will rapidly expand the influence of products in the society, expand the customer base in a short time, and improve brand awareness.

5.4 Brand image promotion strategy

a. Through the cooperation with the government and other enterprises, we can improve product awareness and establish a good brand image.

b. Regularly issue questionnaires, collect and feedback user opinions in time, improve user satisfaction and increase user stickiness.

c. Regularly hold the theme education activities and organize or sponsor various competitions.

6. Business Model

6.1 Main activities

We can provide plasmids and modified bacteria according to customer needs. We can also provide the scientific research teams or other iGEM teams with technical training.

As a widely used technology, it can provide customized services for research, production and other scenarios.

6.2 Profitability

We divide our prices according to bacteriophage and plasmid needs. According to the complexity of specific requirements, we charge 600 yuan - 2000 yuan and other price ranges.

6.3 Cost Structure

Our main costs are laboratory rent, labor cost, publicity cost and material cost of E. coli culture. According to the interview and our investigation, the raw material cost of the bacteria is low, accounting for 15% of the total product cost. The equipment functions required by “strainer” are relatively basic. The equipment cost of midstream laboratories and research institutes are relatively low, especially that the average price of each machine is less than 500000 yuan. In terms of publicity, we will invest a certain amount of advertising online and offline, as well as organizing some competitions, activities or technical exchanges. Therefore, the cost in this regard cannot be ignored.

7. Risk Prediction and Prevention

7.1 Operational risk and avoidance

7.1.1 Operational risks

a. Large investment in production equipment, but the demand from users does not reach a certain ratio to the number of equipment, resulting in idle equipment and no profit;

b. The possibility of failing to make a profit despite policy support because of private enterprises starting late;

c. Customers' requirements may be difficult, and there may be a cost of failure.

7.1.2 Risk avoidance

a. Cooperate with other universities and formulate talent training plans to accumulate long-term strategic cooperation intentions;

b. Develop a reasonable remuneration and benefits system to attract talents and ensure that there is no brain drain;

c. The enterprise shall carry out strategic cooperation with institutions with social welfare nature in order to properly respond to emergencies;

Strengthen corporate culture and improve corporate cohesion.

7.2 Management risks and avoidance

7.2.1 Risks to management

a. Management decisions affect the implementation of the company's strategy and impact revenue and profits;

b. Different cell types and unfamiliarity of staff with cells lead to problems in the experiments.

7.2.2 Risk avoidance

a. Establish sound rules and regulations to clarify the responsibilities of each staff member and to strengthen the supervision of each department;

b. Establish cooperation with professional management bodies to provide the management guidance to the enterprise;

c. Introduce management-related talents to improve the company's decision-making ability.

7.3 Biological risk prediction and prevention

Security is an aspect that we need to focus on. During the operation of the project, we may encounter problems in biosafety and environmental safety. Therefore, we will respond to these problems according to FDA and the Biosafety Law of the People's Republic of China.

"Strainer" can be used for different types of cells. Different cells have different effects on the environment and workers. To minimize this problem, we only choose the cell types allowed in FDA or the Biosafety Law of the People's Republic of China to carry out relevant work.

In terms of biosafety, in order to ensure the normal and safe use of "strainer", we will formulate strict rules and regulations, regularly carry out all-round killing work, carry out relevant safety training for staff, and sign laboratory safety agreements with them; at the learning stage, we will send professionals to guide and maintain communication throughout the process.

As for the environment, our cells may pollute the environment if they leak, so according to the regulatory requirements of FDA or the Biosafety Law of the People's Republic of China, we will seriously consider the site selection of the factory, and the waste generated in the production process will be treated in strict accordance with the regulations to prevent biological pollution caused by waste.

In the later stage, we also consider the technical research and development on the suicide switch, so that bacteria can’t survive without the specific experimental environment, so the application of "strainer" will be safer.