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

Abstract

Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes under the control of a T7 promoter integrated in the genome. We used TRACE in a MEK1 inhibitor-resistance screen, and identified functionally correlated mutations.

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Fig. 1: TRACE enables highly efficient targeted mutagenesis.
Fig. 2: TRACE enables continuous somatic mutations in targeted gene loci and identification of correlated MEK1 inhibitor-resistance mutations.

Data availability

The sequencing data supporting the findings of this study are available in NCBI BioProject database with accession number PRJNA555784.

Code availability

All custom code will be available upon request.

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Acknowledgements

We thank Y. Jiang for help with generating figures, and J. Strecker for purified TN5 transposase. F.C. acknowledges support from Eric and Wendy Schmidt as funders of the Schmidt Fellows Program at the Broad Institute. This work was supported by the NIH Director’s Early Independence Award (DP5-OD024583) to F.C. H.C. is supported by The Lalor Foundation. S.L. is supported by a Molecular Biophysics Training Grant (NIH/National Institute of General Medical Sciences T32 GM008313) and the National Science Foundation (NSF) Graduate Research Fellowship Program. K.G. is supported under Graduate Fellowships from the Fannie and John Hertz Foundation, and the Charles Stark Draper Laboratory. A.L. is supported by a Paul and Daisy Soros Fellowship for New Americans and the NSF Graduate Research Fellowship Program.

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Authors

Contributions

H.C. and F.C. designed experiments. K.G. designed prototype constructs. H.C., S.L., S.P., D.L., and J.L.M. performed experiments. S.L., A.L., and F.C. contributed computational tools. H.C., S.L., D.L., A.L., T.Z., and F.C. analyzed the data. F.C. conceived of and supervised the study. H.C., S.L., and F.C. wrote the manuscript with input from S.P., D.L., K.G., A.L., T.Z., and J.L.M. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Fei Chen.

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Competing interests

Some of the authors (H.C., S.L., S.P., K.G. and F.C.) have filed a patent related to this work.

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Integrated supplementary information

Supplementary Figure 1 Deaminase-T7 RNA polymerase (T7 RNAP) fusion maintains the transcriptional activity of T7 RNAP.

Fusing a cytidine deaminase to T7 RNAP does not significantly hinder the transcriptional activity of the T7 RNAP. Each pEditor variant was introduced into HEK293T cells together with pTarget in which the EGFP gene was solely under the control of a T7 promoter. EGFP signals were observed in cells transfected with pT7, pAPOBEC-T7, pAPOBEC-T7-UGI, pAID-T7, and pAID-T7-UGI, but not in cells transfected with pAPOEBC. The experiments were repeated 3 times with similar results. Scale bar, 200 µm, applies to all micrographs.

Supplementary Figure 2 TRACE demonstrates high editing specificity in human cells.

a. Overexpression of cytidine deaminases alone (pAPOBEC or pAID) in cells result in mutation rates that are not statistically different to mutation rates in the pT7 group (pT7 vs. pAPOBEC, two-sided t test, p=0.9661 in C->T, p=0.8887 in G->A; pT7 vs. pAID, two-sided t test, p=0.2511 in C->T, p=0.1903 in G->A). Bars represent mean ± SEM (N=6 biologically independent cell samples for pT7, 4 for pAPOBEC, and 3 for pAID). b. Mean C->A and G->T (left), C->G and G->C (right) mutation rates in pAID-T7 and pAID-T7-UGI group. Dashed line represents the mean sequencing error rate for the indicated base substitution. Bars are mean ± SEM of N=4 biologically independent cell samples for pAID-T7and pAID-T7-UGI).

Supplementary Figure 3 TRACE induces negligible off-target effects.

a. Experimental workflow on the generation of a sequencing library for off-target analysis. b. Mutation rate per base in target vs. off-target regions of the genome. All bars in the figure are mean ± SEM of N = 6 biologically independent samples for off-target analysis and N=4 for on-target analysis.

Supplementary Figure 4 Mutation profiles upstream, downstream of and at the T7 promoter sequence following TRACE diversification.

a. Mean C->T and G->A mutation rates for the upstream region of the T7 promoter vs. the downstream region. Upstream vs. downstream, two-sided t test, p=0.0363 in C->T, p=0.0205 in G->A for pAID-T7-UGI; p=0.5403 in C->T and p=0.4251 in G->A for pT7; p=0.8983 in C->T, p=0.0861 in G->A for pAID-UGI. b. Mean ->T and ->A mutation rates at each base of the T7 promoter. All bars in the figure are mean ± SEM of N = 3 independent experiments.

Supplementary Figure 5 TRACE induces continuous diversification on barcoded targets.

a. Schematic of two possible models of target diversification by TRACE (continuous diversification vs. one-pulse diversification) which could explain the results shown in Fig. 2b. b. Representative sequencing reads alignments of 18 different barcoded targets. Dendrograms showing the hierarchical relationship of the sequencing reads were constructed using the mutation profile of each read and were displayed on the left of each alignment. Red: A; Green: T; Blue: C; Yellow: G. c. In addition to Fig. 2c, representative sequencing reads alignment of additional 2 unique barcoded targets detected over 3 time points were shown. Dendrograms showing the hierarchical relationship of the sequencing reads across time points were constructed using the mutation profile of each read and were displayed on the left of each alignment. Shared mutations across time points were highlighted in yellow boxes. Red: A; Green: T; Blue: C; Yellow: G.

Supplementary Figure 6 Genome-integrated TRACE induces targeted somatic mutations in genomic loci.

a. Both the editor (pAID-T7) and the T7 promoter-controlled gene (T7 promoter-EBFP) were integrated into the genome of HEK293T cells. The expression of the editor protein was under the control of a doxycycline (dox)-inducible promoter (upper panel). Line plots showing C->T and G->A mutations in targeted gene loci over a period of 20 days in cells harboring the TRACE system (lower panel) starting from Dox induction. Each point is a mean ± SEM of N = 3 independent experiments. b. Cell viability (number of live cells/total number of cells x 100%) with and without dox induction at the indicated time points. Dox vs. no Dox, two-sided t test, p = 0.1911 for day 6, p = 0.3771 for day 10, p = 0.4470 for day 16, p = 0.8675 for day 20. All bars are mean ± SEM of N = 3 independent experiments.

Supplementary Figure 7 TRACE evolves BFP to GFP.

a. TRACE evolves BFP to GFP via a single H66Y amino acid substitution (left) and mutations can be visualized (right). The experiments were repeated 3 times with similar results. Scale bar, 75 μm; insets, 10 μm. b. Ratio of GFP-positive cells to BFP-positive cells. pAID-T7 vs. pAID-UGI, two-sided t test, p=0.0318; pAID-T7-UGI vs. pAID-UGI, two-sided t test, p=0.0441. Bars are the mean ± SEM of N=3 independent experiments. c. Summary counts for the number of BFP-positive cells and GFP-positive cells identified in microscopy images from 3 independent experiments.

Supplementary Figure 8 Mutation profiles of single MEK1 molecules.

Mutation profiles of 93 single MEK1 molecules from trametinib-resistant colonies as identified by Sanger sequencing. Molecules containing both E38K and V211D mutations are highlighted with a red box.

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Supplementary Materials

Supplementary Figures 1–8 and Supplementary Tables 1–6

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Supplementary Data Set 1

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Chen, H., Liu, S., Padula, S. et al. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat Biotechnol 38, 165–168 (2020). https://doi.org/10.1038/s41587-019-0331-8

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