Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor

TRACE (T7 polymerase-driven continuous editing) is a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase.

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Directed evolution, the 2018 Chemistry Nobel Prize winning idea that we can harness evolution by engineering selection, captured our fascination from the beginning of this project. We noted early on the numerous strategies: from phage display [1] (where viruses that infect bacteria cause proteins to be displayed on the surface of cells, enabling screens of large libraries of proteins) to CRISPR-X [2] (where a catalytically inactive dCas9 recruits editors to mutagenize endogenous targets.) In doing so, we began to observe a missing niche; we wanted to design a tool that could enable continuous, targeted mutagenesis in mammalian cells, with potential applications in directed evolution and lineage tracing. 

We approached the problem from a rational engineering perspective (Fig 1). We needed something that was processive and could be targeted to specific sites that we wanted to mutate. T7 RNA polymerase (T7 RNAP), from the T7 bacteriophage, binds to a T7 promoter sequence that is not found in the mammalian genome. Putting the T7 promoter sequence upstream of DNA of interest would enable the T7 RNA polymerase to read through that DNA. As for the editing, we fused a cytidine deaminase (which converts C nucleotides to U nucleotides, which were subsequently read as T nucleotides) to the T7 RNAP, while also recognizing that any base editor could be used to expand the tool’s utility.

Notable strengths over other technologies are the tool’s long editing windows (over~ 2000 bases), continuousness, high mutation rates (~1-4 mutations 1000 kbp over 3 days) with low background editing (no off-targets observed at a detection limit of 10^-6 per bp). These were highlighted in an experiment we did screening for mutations within Mitogen-activated Protein Kinase Kinase 1 (MEK1) that promote resistance to pharmacologic inhibition in mammalian cells. To our surprise, we found a functionally relevant double mutant (E38K V211D). Conventional screening methods by generating mutations within individual mutation libraries would have been prohibitively costly for identifying such correlated mutations. In contrast, when combined with long read sequencing, our tool readily identified correlated mutations.

As for what to call it, we toyed with a few names. We thought TRACE (T7 polymeRAce-driven Continuous Editing) would appropriately capture what we hoped the tool would be used for beyond continuous editing. In our paper, we demonstrate that barcodes continuously mutated by TRACE can be tracked over several generations of cell divisions; as the scientific community’s questions about cell fate grow, we hope that TRACE will be applied for lineage tracing in addition to directed evolution.

[1] Esvelt, K.M., Carlson, J.C. & Liu, D.R. A system for the continuous directed evolution of biomolecules. Nature 472, 499-503 (2011).

[2] Hess, G.T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nature methods 13, 1036-1042 (2016).

Read the paper here.

Chen, H., Liu, S., Padula, S., Lesman, D., Griswold, K., Lin, A., Zhao, T, Marshall, J.L., Chen, F. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat Biotechnol (2019) doi:10.1038/s41587-019-0331-8

Figure 1. T7 RNA Polymerase fused to a cytidine deaminse enables continuous editing.
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Sophia Liu

Graduate Student, Harvard University

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