Team:BNU-China/Implementation
Through lineage tracing, the cell line can be traced and observed to map the cell lineage. Thus, cell lineage tracing plays a vital role in studies related to cell development, differentiation and heterogeneity. In recent years, the method using CRISPR/Cas9 to change barcodes (DNA sequence acting as a genetic marker of single cell) and label different cells becomes popular. However, it has following shortcomings:
1.The constitutively expressed Cas9 consumes barcodes quickly;
2.The total number of barcodes is limited;
These two defects limit the generations it can trace, which even make it impossible to be used in adults.
Considering the importance of lineage tracing and the limitation of existing methods, we designed the ABC system. We used CLB2 promoter and a degradation tag to switch on and off the activity of Cas9, thus coupling Cas9 with cell division and labeling each cell automatically. What’s more, to improve the diversity of barcodes, we used homing guide RNA (hgRNA) to replace the single guide RNA (sgRNA). Considering the practical use, we combined this technology with single cell RNA sequencing to obtain the lineage information out of the transcriptomic information by the double promoter module.
Who and how?
As our project designed a system that can label different cells automatically and show relationships between generations, the main application scenario is in the laboratory for researchers. We plan to construct integrative plasmids containing a cassette that can be used to express and degrade Cas9 periodically, an inducible double promoter cassette and hgRNAs. The function parts will be integrated into the cell's genome. After cell division and differentiation, the researchers can collect samples andcarry out single cell RNA sequencing. Compared with the existing technology, our system has these advantages:
1.Trace the relationships between cells automatically.
2.Trace one and half times more generations and label about eight times more cells.
3.The lineage information can be read out together with transcriptomic information at specific stage or specific tissue.
We interviewed several professors from different fields and they spoke highly of our work and agreed that the ABC system can bring breakthroughs to research like stem cell therapy, embryonic development, organ transplantation, tumor-formation and cell heterogeneity. The advantagesof our system meet the urgent needs of scientists and can solve many problems that puzzle them now.
Examples?
For example, the ABC system can be applied in researches on oncogenesis. Compared with previous methods, the ABC system can trace more generations and label more cells in every generation, which can provide researchers with a more complete map of the development of tumors. What’s more, the ABC system enables the barcodes to be read out at RNA level. By combining the ABC system with single cell RNA sequencing, the lineage information can be obtained together with transcriptomic information (for example, which genes are up-regulated and which genes are down-regulated at that time), which can help researchers understand the molecular mechanism of oncogenesis better.
Apart from tracing the pathological process, the ABC system can also contribute to stem cell therapy. This technology tries to inject stem cells into the diseased organ to renew it, but it doesn’t work so well now. Tracing how stem cells differentiate into organ cells is the major challenge of stem therapy. However, previous cell tracing methods need to be induced artificially which makes it inconvenient when used in living mammals. Therefore, doctors are eager to find easier ways to know what happens to stem cells in the organ. Our ABC system featuring automatic and efficient lineage tracing perfectly meets their needs. It can help them better monitor the differentiation of stem cells and improve stem therapy. Besides these two applications, the ABC system can also apply to research topics like organ transplantation, embryonic development, and so on. All in all, our system can be used widely in many related fields and greatly contribute to medical treatment and scientific research.
Safety?
As researchers involved in biological research, we shoulder the social responsibility. Safety was one of the most important aspects in our project and we considered all factors related to it. The CRISPR/Cas9 system has an inevitable off-target effect and the altered hgRNA may target unexpected genes, which may lead to genetic mutations and bring unpredictable effects to experimental animals. Once leaked into the environment, it may cause gene contamination. Thus, our project is restrained in the laboratory and users need to comply with the basis of laboratory safety standard. We will try to weaken off-target effect of Cas9 from three aspects: rationally designing hgRNA, switching to high-fidelity Cas9, and changing the concentration of Cas9/hgRNA. To avoid altered hgRNAs targeting genome, we hope to set up a registry for suitable hgRNA sequences, which may provide guidance for potential users.
Challenges?
Our project is also facing some challenges. Although we have already verified our project preliminary by different ways in yeast, we still need more experiments to apply our ABC system in mammalian cells. Also, we want to make it more compatible with various species, which requires suitable promoters and hgRNAs. Thus, there is still a long way to go to make our system more mature.
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Email：igem2020bnu_china@163.com
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