Human Practices

1. Problem

In 2020, Prof. Emmanuelle Charpentier from France and Prof. Jennifer Doudna from the United States were awarded the Nobel Prize in Chemistry for developing CRISPR/Cas editing system[1]. As the third-generation gene editing tool, the CRISPR/Cas system works on the basis of simple RNA-guided DNA recognition, and has attracted increasing attention. CRISPR/ Cas system has become the new favorite of gene editing due to its advantages of flexibility, convenience, low cost and high specificity. It has showed broad application prospects in genome engineering, high-throughput functional genome screening, genetic disease treatment, genetic disease diagnosis and other fields[2].

However, the CRISPR/Cas system still has some problems, such as off-target cleaves, variable gRNA efficiency, protospacer-adjacent motif (PAM) specificity, toxicity of Cas protein and other factors, contributing to its limited development[3-5]. During the investigation, we found out that CRISPR-based gene editing tools were inefficient. This made it difficult to screen edited strains accurately, forcing us to increase the number of colonies examined. Especially in the high-throughput field, low efficiency prolongs detection and screening time and costs more manpower and materials. For example, if we want to obtain 10,000 mutant strains using traditional microplate screening methods, the number required for strains screened by the 96-well plate will be "10,000 / editing efficiency". And the time for screening a single strain is 0.5min[6]. For the low efficiency, the unedited strains in the system will increase, which requires more time and labor costs.

Based on the rapid development of the CRISPR technology and the subsequent troubles caused by the low efficiency of the strain construction, there is an urgent need for a gene editing tool with high efficiency and high accuracy to increase the proportion of target strains and reduce the time and cost of the subsequent operations.

References

[1] Zhu, H.; Li, C.; Gao, C., Applications of CRISPR-Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Biol 2020, 21 (11), 661-677.

[2] Wang, S. Y.; Du, Y. C.; Wang, D. X.; Ma, J. Y.; Tang, A. N.; Kong, D. M., Signal amplification and output of CRISPR/Cas-based biosensing systems: A review. Anal Chim Acta 2021, 1185, 338882.

[3] Tan, Y.; Chu, A. H. Y.; Bao, S.; Hoang, D. A.; Kebede, F. T.; Xiong, W.; Ji, M.; Shi, J.; Zheng, Z., Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. Proc Natl Acad Sci U S A 2019, 116 (42), 20969-20976.

[4] Liu, R.; Liang, L.; Freed, E. F.; Gill, R. T., Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing. Trends Biotechnol 2021, 39 (3), 262-273.

[5] Xiang, X.; Corsi, G. I.; Anthon, C.; Qu, K.; Pan, X.; Liang, X.; Han, P.; Dong, Z.; Liu, L.; Zhong, J.; Ma, T.; Wang, J.; Zhang, X.; Jiang, H.; Xu, F.; Liu, X.; Xu, X.; Wang, J.; Yang, H.; Bolund, L.; Church, G. M.; Lin, L.; Gorodkin, J.; Luo, Y., Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning. Nat Commun 2021, 12 (1), 3238.

[6] Zhu X., Construction and application of high-throughput screening platform for industrial microorganisms based on microplate technology and microfluidic technology. East China University of Science and Technology, 2018.

2. Our Human Practices Trip

Solving a problem generally requires three steps: identifying the problem, analyzing the problem, and properly dealing with the problem. In this journey, we need to be reflective, responsive and responsible. Considering our projects from the public, technological innovation, environmental friendliness and other aspects, we tried to make our science to be understood by the society and to reflect its value in the society.

2.1 Designing Our Framework

In the course of our project, we have been working on analyzing the community's problems and feedback to improve our plans and routes, so that our project were carried out successfully and we maximized its value.

Early in the project, we focused on the isopropanol which had a surge in use due to the COVID-19, and interviewed three disinfection companies (Shenzhen LYSOFORM Company, JINFA Disinfect Company and Shandong GUANGWEI Disinfect Company) to investigate the potential market of isopropanol. Through our further literature research and interviews with isopropanol manufacturers, we found that although the isopropanol was widely used, the production mode had problems such as pollution and waste of resources. Based on this, we designed a greener and more sustainable biological production method through high-throughput library construction and directed evolution, to obtain high-yield and high-tolerance strains.

The turning point of the project route appeared after the interview with Yancheng Super Company, which is a leading enterprise in the production of isopropanol. They pointed out that the current production capacity of isopropanol is excessive, half of the manufacturers in China are in a state of suspension and the pollutant discharge cannot meet the national requirements. Through further investigation, we found that isopropanol is indeed in a state of oversupply. Due to the insufficient development of downstream industries and the good control of the epidemic, the export volume of isopropanol fell by more than 50% year-on-year in 2021. At the same time, the maximum titer of isopropanol produced by the biological method is 17.5g/L[1], which is difficult to reach the production level of the chemical methods. And during the interview, enterprise personnel said that the production of isopropanol by the chemical method tends to be sophisticated, and the promotion of the biological methods, such as the large-scale fermentation, is difficult.

Reference

[1] Liang, L.; Liu, R.; Garst, A. D.; Thomas, L.; Violeta, S. i. N.; Gregg, T. B.; Ryan, T. G., CRISPR enAbled trackable genome engineering for isopropanol production in Escherichia coli. Metab Eng 2017, 41, 1-10.

With the in-depth investigation of the production status of isopropanol, we come to the following conclusions:

1) Isopropanol is an excellent disinfectant, but the public and users do not know it well, and has low level of recognition.

2) Isopropanol is mainly produced by the chemical method at present, but there are problems like serious pollution, high cost of raw materials and equipment maintenance.

3) Biological preparation of isopropanol is more friendly to the environment, and the biological method is not dependent on the raw material of acetone, which is conducive to saving oil resources. However, this method still has low-yielding problem in laboratory, and is hard to be applied to industrial production.

Strain construction is the core of the biofabrication and synthetic biology. Strain design and strain screening are two key steps. Gene editing technology provides a convenient tool for strain design and modification. However, in our investigation, we found that the efficiency of gene editing fluctuated during strain construction, which would lead to complicated screening procedures, especially in high-throughput fields. Therefore, we further focused our project objectives on improving the efficiency and accuracy of the editing tools, thereby simplifying the subsequent screening and sequencing operations, saving time and reducing cost. It can not only be applied to the field of biological fermentation, but also play a role in more fields involving gene editing, and provide a convenient and efficient tool for the synthetic biology community.

We interviewed experts and enterprises, engaged stakeholders in the communication of understanding and being understood, and actively engaged in dialogue with biosafety and environmental protection organizations at the same time. We hope that our program will bring a beneficial and creative change to the synthetic biology community.

2.2 Public Perception

The public perception in the community is an important reference and guide for the design of our project. Therefore, we designed a questionnaire for students, participating teams and people working in the field of gene editing. We want to explore the current public awareness and recommendations in the field of gene editing and high-throughput.

We received more than 190 answer sheets and the results are shown below:

The CRISPR/Cas9 system we used is known or heard of by 58.2% of the respondents. They pointed out several problems and disadvantages of the CRISPR/Cas9 system, such as high false positives, off-target cleaves and limited application. At the same time, 99% of those who know the system thought that the editing efficiency and stability need to be improved. In addition, some respondents who know about high-throughput screening claimed it was important for the field of synthetic biology, and 95% of them thought that high-throughput screening was low-efficiency. The survey population all expressed the need to improve the efficiency of the gene editing tools.

In the survey, we also found that most people are not familiar with the high-throughput screening technology, and 50.2% of the respondents have never heard of it before. In conclusion, we think it's important to propagandize and introduce the CRISPR/Cas9 system and high-throughput screening technology to the public. At the same time, we also believe that such work will make a valuable contribution to the field of synthetic biology.

1、 Do you know the CRISPR/Cas 9 system?

The CRISPR/Cas9 system we used is known or heard of by 58.2% of the respondents.

2、 Do you think it is necessary to improve its editing efficiency and stability as a gene editing tool?

After seeing that over 90% of researchers claimed that it was necessary to improve the editing efficiency and stability, we deeply believed that we had to work hard on upgrading the gene editing tool.

3、 Do you know about high-throughput screening technology?

We also found that most people are not familiar with the high-throughput screening technology, and 50.2% of the respondents have never heard of it before. So we believe that we need to make a good dissemination of high-throughput screening technology.

4、 Do you think there is a need to improve the efficiency of high-throughput screening?

Same as the attitude to the improvement of the gene editing tools, there are still some researchers wanting to improve the efficiency of the high-throughput screening.

5、 Do you think that improvements in the efficiency of gene editing are critical to the field of synthetic biology?

We were shocked by the ratio, since there is only 4%. However, after deeper statistics, we found out it is the one who didn’t know about CRISPR or high-throughput screening voted that it is not critical at all.

2.3 Be Socially Responsible

Synthetic biology has infinite potential and possibilities, as well as great application prospects, so how to ensure that the innovation of synthetic biology can truly benefit society without negative effects is a key issue in this field. Our project will focus on "doing the right thing" and "making things right" to improve our program in biosafety, security, social ethics, environmental friendliness and other aspects.

We participated in the CCiC Biosafety and Human Practice series and discussed project safety issues with Liu Yanfeng, the CCiC Executive Committee member, Xu Can, the Secretariat member, and other iGEMers in China. They emphasized that when comes to the applications in the field of gene editing, the first thing should be considered is the legal judgment, such as discussion of the application scenarios for our technology, the boundaries of its use, and the possibility of justifying it. At the same time, biosafety has to be considered when applying our technology to biological production. They proposed that if we apply this technology to the directed evolution of strains and the production of strains, we should consider the genetic contamination and environmental pollution caused by the strain leakage. We were also advised to be more aware of safety in practice and to eliminate the risk of contamination by leakage.

Professor Fan of Dalian University of Technology has nearly a decade of experience in leading UN simulations and has a deep understanding of various UN documents and contexts.

At the beginning of the project, we discussed with Professor Fan the issues that need to be paid attention to in the design. Professor Fan proposed that we should have a deep understanding of the purpose and significance of the project itself. The problem to be considered should be the key point as well as the social concern, which needs further in-depth discussion and interrogation. The biosafety issues involved cannot be ignored, especially the strain modification, which might cause contaminate the environment and damage the human body. The biosafety protections of the Sustainable Development Goals and the relevant United Nations documents on biosafety have paid attention to the related issues.

Takeaways: We should have a deeper understanding of the connection between the Sustainable Development Goals and the iGEM community and clarify the responsibility and feelings of the sustainability concept. And we try to make our project more sustainable, safer in biology and take more responsibility in the society.

2.4 Communication & Education

Popularization of science is an important and direct way for people to understand science. Synthetic biology, as a "life science weapon", is very essential to break the barrier between it and public. Our team created a fantastic song to bring synthetic biology to the public through a popular format. We published several articles on WeChat official account to introduce synthetic biology and its related fields to the public. Simultaneously, we also publicized the application of isopropanol to increase public awareness, help and pave the way for its future downstream industry expansion and the overcapacity problems.

At the same time, we are highly aware that the power of one person is small, so we sought partners and helped each other. We have formed a publicity team with Ocean University of China and Northeastern University to enlighten the primary and secondary school student groups on biological education. What’s more, we have involved in writing the "Synthetic Biology Guidebook" edited by Jilin University with several universities, sharing our experience in work and team formation with future iGEMers.

3. Our Closed Loop

We started from the synthetic biology community and collected the current problems of CRISPR by interviewing companies and experts and distributing questionnaires. During the project, we actively communicated with stakeholders to improve our project with reflection, feedback, and responsibility to make it more feasible, practical, and safer. Later, we gave back to the community to solve the problems of the editing tools and further extended the project to the industry. At the same time, we used a combination of online and offline forms to educate the public about science, improved the public's knowledge of synthetic biology and CRISPR, and promoted isopropanol to pave the way for the future industrial development of the project.

Integrated Human Practices

Phase 1 - Problem Finding

At the beginning of the project, we conducted research through literature reviews, laboratory visits, and corporate interviews, finding that the current gene editing efficiency fluctuates, while high-throughput gene editing efficiency ranges from 60% to 98% during library construction [1-3]. Besides, the editing efficiency of such method is variable when targeting different locus of genome, leading to tedious and time-consuming follow-up operations.

We interviewed the teachers and fellow graduate students in Prof. Zhiwei Zhu's group at the School of Biological Engineering, Dalian University of Technology, which is currently using the CRISPR/Cas9 system for sequential editing of E. coli, and we desired to find out if there were any problems in its experimental process.

Takeaways: Through the conversation, we learned that as the experiment proceeded, the efficiency decreased significantly after multiple consecutive rounds of modification on the same genome, and the picked strains were all false positives, which seriously affected the subsequent operation and the experimental process.

Takeaways: We started a conversation with Mr. Du from Bluepha and learned that CRISPR/Cas9 has different editing efficiency for different chassis strains, and it also has efficiency fluctuation problem when editing. In the high-throughput field, the tedious operation of screening assays has been always the bottleneck. During the screening, if you want to get a certain number of target strains, you need to calculate it with the minimum efficiency.

Based on the above investigations, we assumed three experimental scenarios, which are large fragment editing, high-throughput library construction, and fragment editing after library construction, then we extrapolated and compared the time and screening cost at different efficiencies.

We hereby assume that:

10,000 mutant strains, editing efficiency of , metabolic pathway length of 5,000 bp [4-8], single sequencing price of $20, 96-well plate for one sample set to 0.5 min, average monthly salary set to 10,000 yuan, 8 hours of work per day, 30 days of work per month. In addition, for the price of 96-well plates, we obtained the prices of ten different merchants and took the average value to substitute for the calculation.

Table1. Prices of 96-well plates sold by different ven dors (source of quotation: internet/telephone inquiry, unit price: RMB)

Calculation formulas:

Sequencing price cost:

Screening time:

Labor cost:

Scenario 1: Editing of large fragments

Cost:

Note: 5000bp is the average metabolic path length of the surveyed literature, 500bp is the single sequencing length.

Scenario 2: Construction of high-throughput libraries

Time：

Cost：

Note: 0.5min is the screening time for individual strains in microplates. Same below.

Scenario 3: Constructing a high-throughput library and then editing large fragments

Time：

Cost：

According to the above survey and analysis, we can tell that under the incomplete cost statistics, the improvement of efficiency will simplify the subsequent operation, which is necessary for the cost and time saving. The efficiency of the current high-throughput editing tools is not well stabilized in the high range, which makes the subsequent screening and sequencing operations difficult. Therefore, we would like to design a tool to improve the proportion of the edited strains and simplify the subsequent screening and sequencing steps and costs.

References

[1] Liu, R.; Liang, L.; Choudhury, A., Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production. Metab Eng 2018, 47, 303-313.

[2] Wang, H. H.; Isaacs, F. J.; Carr, P. A., Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460(7257), 894-898.

[3] Liu, R.; Liang, L.; Choudhury, A., Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production. Metab Eng 2019, 51,50-58.

[4] Lu, L. Y.; Shen, X. L.; Sun, X. X.; Yan, Y. J.; Wang, J.; Yuan, Q. P., CRISPR based metabolic engineering in non-model microorganisms. Current Opinion in Biotechnology 2022, 75, 102698.

[5] Lei, Y.; Lu, L.; Liu, H. Y.; Li, S.; Xing, F.; Chen, L. L., CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants. Molecular Plant 2014, 1494-1496

[6] Dina Simkin; Vasileios Papakis; Bernabe I. Bustos; Christina M. Ambrosi; Steven J. Ryan; Valeriya Baru; Luis A. Williams; Graham T. Dempsey; Owen B. McManus; John E. Landers; Steven J. Lubbe; Alfred L. George, Jr; Evangelos Kiskinis. Homozygous might be hemizygous: CRISPR Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls. Stem Cell Reports 2022, 993-1008.

[7] Tara Moorea; Kathleen A. Christiea; John Marshallc; M. Andrew Nesbita, Personalised genome editing-The future for corneal dystrophies. Progress in Retinal and Eye Research 2018, 65, 147-165.

[8] Qin, W. F.; Xia, Y. J.; Xiong, Z. Q.; Song, X.; Ai, L. Z.; Wang, G. Q., The intestinal colonization of Lactiplantibacillus plantarum AR113 is influenced by its mucins and intestinal environment. Food Research International 2022, 157，11382.

Phase 2 - What We Can Do

In this phase, we designed the project and tried to make it more reasonable and feasible.

Dr. Yu Zhang believed that we can trigger initiation by line step by step, which was overall reasonable and feasible. She proposed that we need to consider whether the plasmid of the toxic gene was single copy or not, and whether there would be the possibility of editing a toxic gene and still having other unedited copies leading to strain death. We also needed to consider the toxicity of Cas9 protein. Based on Dr. Yu Zhang's suggestions, we reviewed our project design.

We set up a reverse screen while constituting the toxic gene as a plasmid alone to achieve a dual screen of exerting toxic/plasmid excess death, resulting in an increased proportion of the target strains. The plasmid containing Cas9 was transformed into a medium copy to reduce the damage to the strain.

We discussed the project idea with Dr. Qü, who suggested that we could use the induction system to make the plasmid contain SOS promoter to achieve the effect of successful editing and detoxification due to the toxic gene. At the same time, we questioned the effect of the lower copy of Cas protein on the editing efficiency. Dr. Qü considered that the editing efficiency was not high because of not only the problem of Cas9, but also the problem of gRNA activity and expression amount. As long as Cas9 can be continuously expressed, the editing efficiency can be increased by reasonable design of gRNA, as well as increasing the gRNA expression amount and extending the incubation time.

Our project serves the synthetic biology community, so we want our design to be more in line with the specifications and more feasible. We talked with Prof. Zhu, an expert in synthetic biology, who thought our design was feasible, reasonable and significant. He also suggested that SOS should be able to initiate the gRNA that targets the toxic gene, and the promoter of SOS response is the key to our success.

We exchanged the project ideas with Mr. Liu. Since our project requires the introduction of three plasmids into E. coli at the same time, Mr. Liu recommended us four compatible expression plasmids in E. coli BL21: high copy (pRSFDuet-1), relatively high copy (pETDuet-1), medium (pCDFDuet-1), and weak (pACYCDuet-1) plasmids. He also suggested us having a deeper understanding of ori and resistance genes on it; and recommended the commonly used inducible promoters pT7, pTac, pTrc. Mr. Liu also recommended methods for screening E. coli phenotypes such as antibiotics, fluorescence intensity or nutrient deficiency.

Phase 3 - Reflection and Response

Since the purification system we designed contains two gRNAs on the same plasmid, we discussed the issue with Prof. Wu in view of the interaction between the two gRNAs. Prof. Wu suggested that according to the experience of constructing gRNAs for mammalian expression systems, there was no difference between dual-gRNA expression and single-gRNA expression. Each gRNA with its own promoter would not interfere with each other's expression, and also the expression of both gRNAs could be controlled by some promoter element optimization.

In order to avoid the problem that the toxic gene is excessively toxic and affects the normal physiological activities of the strain during the experiment, we asked Dr. Liu for advice. Dr. Liu said that the focus of controlled expression of toxic genes was not on the genes, but on the rigor of the promoter that expressed the toxic genes. To address this issue, he suggested that the highly toxic protein could be partitioned using an intrinsic peptide to make it less toxic when leaked for expression, but revert to strong toxic when expressed. Meanwhile, Dr. Liu recommended us the reference Engineered toxin-intein antimicrobials for our further study.

Prof. Zhu was very interested in our project, and we had been in close contact with him. Therefore, when we encountered the problem of high toxic genes, we also consulted Prof. Zhu at once. Prof. Zhu suggested that there are different solutions for different toxic genes. Overall, it can be achieved by weak promoter. We also talked with other members of Prof. Zhu's group and got suggestions: replace the multi-copy plasmid with a low-copy or single-copy plasmid, or put the toxic protein gene in a backward position when the system has multiple genes to be expressed under one promoter.

During the experiment, when IPTG was used to induce the expression of sacB gene, we were not sure how to design a more reasonable range of IPTG concentration, so we approached Mr. Liu for advice. Mr. Liu shared his experience with us: if the protein expressed by IPTG is a pathway enzyme of the metabolic pathway in vivo, then the amount of IPTG can be less. The amount to be added when doing in vitro purification is 0.1-1mM, but in vivo pathway enzyme expression only needed to add 0.01-0.1mM, the specific concentration can be optimized. If the induced expression of IPTG is still too high, we can change the host, instead of using BL21, you can choose MG1655 (DE3) or JM109 (DE3).

At the same time, we also encountered the problem that some proteins were very toxic and it was difficult to diminish the effect of these proteins on the whole system by reducing the expression of the proteins, for which Mr. Liu gave his suggestions.

1) Firstly, we should understand what causes the strong toxicity of these proteins. Mechanisms such as disruption of metabolic pathways and the occupation of cell membrane channels should be considered. For different strategies, co-expression of chaperone attenuated proteins, cell directed evolution and other strategies can be chosen.

2) The expression of toxic proteins can be induced by considering product-inducible promoters or quorum sensing promoters, so that the enzyme will not be expressed during the growth phase or the expression is relatively low, and only expressed when it enters the stable phase or production phase, which can reduce the impact of protein toxicity on the physiology of cells.

3) A protein degradation circuit can be designed to induce protein degradation at a certain stage.

These suggestions are very instructive. We decide to choose strain MG1655 as the host. And we decide to adjust the expression level of toxic genes by difference strategies, such as using different amount of IPTG to induce gene expression, or to mutant these genes to lower their toxicity.

We chose two toxic genes, ccdB and sacB, at the beginning of our experiment. Since ccdB was still too toxic after modification, the modification of the SacB became the key to our project. We approached Mr. Feng with the problem, hoping to find a way to modify the SacB to reduce toxicity with his assistance. Mr. Feng guided us to add three new sites and recommended the papers Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation (Glycobiology, 2017) and Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis (Biochemical J., 1991). He also advised us to conduct protein mutagenesis experiments and provided technical and equipment support.

Phase 4 - Social and Responsible

In this phase, we actively communicated with experts and multiple companies to make our project more practical and able to play the biggest role in the society as possible.

Professor Fan of Dalian University of Technology has nearly a decade of experience in leading UN simulations and has a deep understanding of various UN documents and contexts.

At the mature stage of the project, we contacted Prof. Fan again and presented her with a more specific refinement of the meaning of the project and the possible issues involved. Professor Fan thought that our project was much better compared to the initial stage, and advised us to keep focusing on the biosafety literature and laws and regulations. Based on the proposal, we studied http://dx.doi.org/10.16418/j.issn.1000-3045.20200206002 and several other related literatures.

Prof. Yuan has an in-depth study on biomass energy production and strain construction, and has rich experience in alcohol fermentation. Her research group produces a variety of products such as ethanol and butanol, so we actively talked with Prof. Yuan to explore the key points that need to be paid attention to in the production of alcohol fermentation. Prof. Yuan proposed that special attention should be paid to the fermentation type of strains during the fermentation. At the same time, we need to pay attention to the toxicity of the isopropanol product, the possible damage to the cytomembrane and the tolerance concentration of the strains. We could cultivate the strains before we took them into production, since when the strain density reached the level of quorum sensing, it may be conducive to fermentation production. It could not be ignored that when we were using bacteria for fermentation, we needed to pay attention to centralized sterilization after fermentation to avoid environmental pollution.