Team:BNU-China/Human Practices
In the field of the molecular mechanism of basic biological processes, cell lineage is crucial. Many researchers have tried to improve the lineage tracing technology for years. Compared with traditional methods, the combination of CRISPR/Cas9 and DNA barcodes has a bright prospect in this field, but the generations it can trace are limited and this problem troubles the researchers. Our team aims to prolong the tracing generation and label more cells. Considering that this technology is generally used in labs, we interviewed researchers in relevant fields. The interview gave professional opinions on the value and feasibility of our project so that we could refine our design at all stages. Through this process, we ensured that our design will have a better application in practice, making our project more targeted, effective and influential.
Problem Investigation
Junjiu Huang, Professor, doctoral supervisor, School of life
sciences, Sun Yat-sen University
Regulatory mechanism of cell senescence and regeneration ;
research and application of cell gene editing technology ;
mechanism of mammalian early embryonic development; etc.
Jiekai Chen, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences/ Principal Investigator;
Research Areas：To study the molecular regulation mechanism of cell fate determination
based on somatic cell reprogramming and to develop a technology system focusing
on the application of induced pluripotent stem cells (iPSCs); to excavate the important
biological mechanism by bioinformatics based on the data of epigenetic group and
single-cell transcriptome collected in the cell fate transformation model; etc.
Reasons for Contact
We need to understand how to conduct lineage tracing with CRISPR/Cas9 and DNA barcodes clearly. What theis current situation of this technology? Can it be further improved in some aspects? What has already been done to improve it? In order to get a more comprehensive view, we contacted Professor Junjiu Huang and Professor Jiekai Chen.
Answers for Questions
We reached out to Professor Huang. He gave us insight into some current status of the technology for lineage tracing. We discussed the existing problems and he stressed that the diversity of barcodes is the main limitation of this technology. There is indeed a long way to go before meeting the needs of lineage reconstruction.
We also consulted Professor Jiekai Chen. He has extensive experience in lineage tracing and we discussed the advantages of CRISPR/Cas9 technology together. The discussion made us realize that the technology has two main problems. First, the diversity of barcodes needs to be further improved; second, the generation that could be traced is limited, and it needs to be regulated by special factors in specific situations. Professor Chen also mentioned the CARLIN system reported on Cell.
Improvements for Project
After obtaining feedback, we put forward our solutions preliminarily. First, we aim to prolong the time of lineage tracing, so we couple cell division with Cas9 to make cells automatically switch on or off the system. Second, we hope to change the base sequence of target sites to increase barcode diversity.
Return for Visit
Later with more literature to support our ideas, we approached Professor Huang again to discuss our newest design, he agreed with us that the improved complexity will contribute to performing lineage tracing more accurately and gave us his best wishes.
Professor Chen told us that combining Cas9 with cell cycle may prolong the labeling time by reducing the cutting frequency of Cas9 during each cell cycle.
Idea Assessment
Jie Na, Principal Investigator and Associate Professor,
Center for Stem Cell Biology, School of Medicine,
Tsinghua University, Beijing, China
Research Fields & Emphasis: Pluripotent Stem Cells, Cardiovascular cell differentiation,
Epigenetics, Regenerative medicine, Mammalian embryo development, Organoid, 3D Bioprinting.
Reasons for Contact
Does our idea make sense? Is it responsible and good for the world？Does it have any potential risks to the world? Professor Jie Na has made great achievements in the study of stem cell differentiation and embryonic development. We got in touch with her by e-mail.
Answers for Questions
Knowing our preliminary idea, she believed that it is innovative and significant to couple Cas9 with cell division. She also mentioned that the application of this design will promote the development of some cutting-edge researches like tracing the lineage of stem cells. We also asked whether mice with random mutations carry risks when using CRISPR/Cas9. She emphasized that any gene-editing experiment should comply with bio-safety laws. The purposes, contents and methods of the experiments should be documented according to the regulations. Experiments can be conducted only after getting permission. Therefore, if the technology is properly used and the mice are treated correctly, this technology will not harm society.
Improvements for Project
The communication makes us more convinced that our project is meaningful. We further consulted literature and investigated the application fields of lineaging tracing technology. At the same time, we strictly standardized the experimental operation and carried out safety training for the team members.
Project Design
Chunlai Chen, Principal Investigator; School of Life Sciences;
Tsinghua-Peking Joint Center for Life Sciences;
Beijing Advanced Innovation Center for Structural Biology, Tsinghua University
Research Fields: Develop cutting-edge single molecule fluorescence methods and techniques;
molecular mechanisms of the ribosome and associated proteins during protein synthesis; etc.
Zhen Xie, Associate Professor; Department of Automation, Tsinghua University,
Center for Synthetic & Systems Biology ,Beijing National Research Center
For Information Science And Technology
Research Fields:Construction of parts and motifs for synthetic biology research
Automation for gene synthesis and genetic circuit assembly
Genetic circuit engineering for gene therapy and cell-based therapy
Integrative biomolecular sensing and processing for cellular automation
1. Build a periodic expression module of Cas9:
Reasons for Contact
Now, it is time to design our project! In terms of coupling Cas9 with cell division, our team put forward various possible solutions. There are two relatively feasible methods. One is to produce luciferase endogenously, using the fluorescence to trigger the binding of two parts of Cas9 (Figure 1). The other is to design a cassette that can periodically express and degrade Cas9.
(Figure 1. Nihongaki et al., 2020)
Answers for Questions
For the first method to couple with Cas9 with cell cycle, we communicated with Professor Chen. He doubted whether the wavelength and intensity of blue light could meet the needs.
Improvements for Project
We thought it was a crucial problem, so we tried to find a suitable luciferase. We found a study done by
Schipper and co-workers on the Fluc-luciferin system for PDT. In this work, however, when luciferin concentration was 20 μM,
the photon output reached only 2.3×103 photons per second per cell, which approximately corresponds to only 1.2×10-9
mW/cm−2. By comparison, the wavelength we need is about 0.12mW/cm2, which is much higher than what
has been reported. We speculated that a very high concentration of luciferin is required to achieve a suitable intensity.
Having identified and understood multiple adverse aspects of using fluorescence, we decided to periodically
express and degrade Cas9. We coupled Cas9 with cell division to achieve its periodic expression and use a degradation tag to
degrade it at a proper rate.
2. Degrade Cas9 at a proper speed:
Reasons for Contact
For our degradation system, we put forward two methods: adding the D-box degradation tag or using Cyclin B2 to degrade Cas9. Due to the influence of epidemic, we didn't have enough time to compare these two methods by experiment. Considering Professor Zhen Xie has rich experience in synthetic biology automation system, we got in touch with Mr. Xie. We held an online meeting with him and asked for advice to help us design better.
Answers for Questions
Professor Xie pointed out that most studies constructed a fusion expression system of Cas9 and an endogenous protein. The half-life of Cas9 would be reduced greatly due to the degradation of the endogenous protein.
Improvements for Project
We thought Cyclin B2 can couple the activity of Cas9 with the cell cycle more tightly, so we chose this method.
3. Increase the tracing number of cells:
Reasons for Contact
In the previous interviews, many professors stressed the importance of improving the diversity of barcodes. Higher diversity can establish a more accurate pedigree tree. We discussed this problem with Professor Xie and put forward our solution: changing the base sequence of the barcode to increase its diversity.
Answers for Questions
Professor Xie told us that after several rounds of cutting, barcodes change too much to match with single guide RNA (sgRNA). Then cutting could no longer happen and barcodes won’t change again. So even if we change the barcode sequence, the improvement is still limited.
Improvements for Project
This feedback inspired us that if Cas9 could recognize and cut sgRNA's own expression box, the cutting will happen continuously.
Then we made further efforts to modify and improve the design. Finally, we surprisingly found that the homing guide RNA (hgRNA) could cut its own expression box! Its diversity is improved a lot when compared with sgRNA, so we decided to use it.
Further Improvement
Guangdun Peng, Principal Investigator & Professor, Guangzhou
Institutes of Biomedicine and Health, Chinese Academy of Sciences
Research Fields: Implementing cutting-edge technologies to investigate
the fundamental mechanisms of stem cell lineage segregation and
promoting the translational application of functional stem cells.
Reasons for Contact
Comparing hgRNA with sgRNA, we found that although hgRNA improve barcode diversity, its information could only be read at DNA level while sgRNA could be read at both DNA and RNA level. Is it necessary to read lineage information at RNA level? So we held an online meeting with Professor Guangdun Peng and raised our concerns.
Answers for Questions
Professor Peng said that lineage tracing described the relationship between cells, while RNA sequencing described cells' states. Their combination enable us to read the barcodes and transcriptome status at the same time, so both the development and type of the cells can be obtained together.
Improvements for Project
We were aware that it is important to read the lineage information out of the transcriptomic information. We designed a constitutive double promoter module and an inducible double promoter module. That brings our project closer to the actual application.
Return for Visit
We contacted Professor Peng again to discuss our project. Professor Peng thought that our design is feasible and rational. In addition, Professor Peng also gave his suggestions on experiments and other aspects. He also pointed out that our design has the potential for practical applications definitely.He believed that our double promoter module did enable the combination of lineage information and transcriptomic information. He suggested us to improve our project further. He believed that our system has promising prospects and will promote researches on cell development.
The exchange with Professor Peng Guangdun made us think more broadly, and it impacted and improved our project profoundly.
Reasons for Contact
We were aware that it is important to read the lineage information out of the transcriptomic information. So we wanted to make it possible. However, the problem is how to express hgRNA and how to read its information.
In order to make our system better serve laboratories engaged in related research, we interviewed stakeholders, such as some companies. What we want to gain from the interview is:
1.To gather insights on the perception of our technology in each application
2.To understand the needs and motivations of our stakeholders
3.To understand how our technology can better serve our stakeholders for each application.
Answers for Questions
Through communication with many biotechnology companies, we learned that the 10X Genomics platform is currently the most commonly used transcriptome sequencing platform, which can complete processes such as single cell isolation, mRNA capture, and reverse transcription. In order to avoid the contamination caused by rRNA, the 10X Genomics system used primers with oligo dT to capture and amplify mRNA.
We learnt that 10X Genomics depends on the polyA tail to read the transcriptomic information. However, previous literature showed that additional bases at the 5’ end of hgRNA may abolish its ability.
Improvements for Project
To solve this problem, we designed a double promoter module so that the lineage tracing information can be read out of the transcriptomic information.
*Achievement*
Sliver
We interviewed several professors, three quarters of them mentioned that our project will have a far-ranging impact on characterizing lineages in development, regeneration, cellular reprogramming, and disease states.
We interviewed Professor Jiekai Chen by email, and he pointed out that researches based on lineage tracing were limited by the marked generations by barcode, which supported the background of our project and emphasized the significance of our project.
We interviewed Associate Professor Jie Na by email to learn about the safety and ethical issues that may be involved in this technology. She also confirmed that it is innovative and significant to couple Cas9 with cell division in lineage tracing technology.
We contacted researcher Jiekai Chen again. He said that combining Cas9 with the cell cycle may reduce the cutting frequency of Cas9 in each cell cycle, thus prolonging the tracing time, which supports the feasibility of our idea.
Gold
Through the communicating with Professor Junjiu Huang, we learned about the background of this technology and discovered the problem that barcode diversity is limited. After reviewing some literature, we chose to use hgRNA to solve this problem.
The CARLIN system mentioned by Professor Jiekai Chen inspired us to regulate the expression of Cas9 to extend the lineage tracing generation, so we designed a system to couple Cas9 with cell division.
Professor Chunlai Chen raised questions about inducing the combination of two fragments of Cas9 by fluorescence, which made us discover the potential problem of this design. So we chose to periodically express and degrade Cas9.
Inspired by Professor Zhen Xie, we discovered that hgRNA can continuously mutate and recognize new barcode sequences. So we used it to improve the diversity.
Professor Guangdun Peng pointed out that it is necessary to read the lineage tracing information at RNA level, so we decided to drive the transcription of hgRNA by two different promoters.
References
[1] Schipper, M. L., Patel, M. R., & Gambhir, S. S. (2006). Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells. Molecular imaging and biology, 8(4), 218–225.
[2] Nihongaki, Y., Kawano, F., Nakajima, T., & Sato, M. (2015).Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nature Biotechnology, 33(7), 755–760.
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