team

The Problem

Early cell lineage tracing techniques used dyes or fluorescent markers which helped resolve major questions in cell development. However, the low number of clones analyzed at any one time point limited comprehensive understanding of global stem cell dynamics within tissues.

In recent years, the method combining CRISPR/Cas9 and barcode, a DNA sequence as genetic marker, has drawn great attention[1]. However, the total number of barcodes is limited and the constitutively expressed Cas9 consumes barcodes quickly. These make barcodes inadequate for the tracing purpose after several generations, giving a biased view on organ complexity and puzzling scientists in this field.

Our Inspiration

An article published in Cell on June 17, 2020[2] established an inducible lineage tracing mouse model based on CRISPR/Cas9 (as shown in the figure1 below). However, the induction of cas9 still needs the addition of exogenous substances and has not yet reached automation.

Figure 1. Schematic of inducible CARLIN system (Bowling, S et al., 2020)

Our Solution

Inspired, we designed the Automatic Barcode Creator (ABC) system.
We built an inducible expression module of Cas9 in association with cell division to label each cell automatically. What’s more, to solve the problem about a limited number of barcodes, we used homing guide RNA (hgRNA) to replace the single guide RNA (sgRNA). Different from sgRNA, hgRNA directs Cas9: hgRNA complex to its own locus, thus simplifying the system by using hgDNA as the barcode and enabling retargeting and evolvability of barcodes.

Further improvement

Through literature review and communication with experts in related fields, we learned that lineage tracing describes the relationships between cells, while single cell RNA sequencing gives information on gene expression at a particular moment. The combination of them is significant. However, the hgRNA expressed by U6 promoter can’t be captured by 10X Genomics platform for lack of polyA tail, making it impossible to read barcodes together with transcriptomic information.
To solve this problem, we designed the double promoter module. U6 promoter is used to transcribe hgRNA, and the promoter in front of it will add the polyA tail, which allows barcodes to be read at RNA level.

The desired effect

  When cells start to divide at the end of G2 phase, Cas9 is expressed under the control of cyclin B2 promoter[3,4] and bound with hgRNA, which guides Cas9 to cut barcodes. Then at G1 phase in the next cell cycle, Cas9 stops expressing and cells perform non-homologous end joining(NHEJ) and introduce random mutations, generating new barcodes. The degradation tag at the N-terminal of Cas9 makes Cas9 degrade quickly[5]. After cell division, Cas9 disappears and no longer cuts the barcode until the next round of cell division.
  When conducting single cell RNA sequencing, the double promoter module we designed transcribes hgRNA and adds the polyA tail, thus enables the lineage information to be read together with transcriptomic information.
  In conclusion, our ABC system can label and trace single cell automatically in a population after many generations and enables the lineage information to be read together with transcriptomic information.

References

[1] Spanjaard, B., Hu, B., Mitic, N., Olivares-Chauvet, P., Janjuha, S., Ninov, N., & Junker, J. P. (2018). Simultaneous lineage tracing and cell-type identification using CRISPR-Cas9-induced genetic scars. Nature biotechnology, 36(5), 469–473.
[2] Bowling, S., Sritharan, D., Osorio, F. G., Nguyen, M., Cheung, P., Rodriguez-Fraticelli, A., Patel, S., Yuan, W. C., Fujiwara, Y., Li, B. E., Orkin, S. H., Hormoz, S., & Camargo, F. D. (2020). An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells. Cell, 181(6), 1410–1422.e27.
[3] Eluère, R., Offner, N., Varlet, I., Motteux, O., Signon, L., Picard, A., Bailly, E., & Simon, M. N. (2007). Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 in S. cerevisiae. Journal of cell science, 120(Pt 4), 702–711.
[4] Wäsch, R., & Cross, F. R. (2002). APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit. Nature, 418(6897), 556–562.
[5] Hendrickson, C., Meyn, M. A., 3rd, Morabito, L., & Holloway, S. L. (2001). The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition. Current biology : CB, 11(22), 1781–1787.

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