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.

Fig. 1 |. TRACE enables highly efficient targeted mutagenesis.

a, Schematic of TRACE. The recombinant fusion of cytidine deaminase and T7 RNAP recognizes a T7 promoter inserted upstream of a target gene. As the T7 RNAP transcribes DNA, the deaminase introduces point mutations (red star).b, Constructs tested in c–e. c, Representative high-throughput sequencing reads after diversification. C>T (green) and G>A (red) mutations are highlighted. d, Mean C>T and G>A mutation rate of the target region. Dashed line, mean sequencing error rate. e, Mutation rate per base across an ~4-kb region. Vertical dashed line, end position of T7 promoter. Horizontal dashed line, mean sequencing error rate. f, Tested T7 RNAP mutations (top) and their effect on mutation rate (bottom). Dashed line, mean sequencing error rate. All bars in the figure (d and f) represent mean ± s.e.m., n = 3 independent experiments. NLS, nuclear localization signal.

Fig. 2 |. TRACE enables continuous somatic mutations in targeted gene loci and identification of correlated MEK1 inhibitor-resistance mutations.

a, C>T and G>A mutations over 20 d with integrated T7 promoter-target (mean ± s.e.m., n = 3 independent experiments). b, Top, distribution of the number of mutations per contiguous read over 20 d. Bottom, mean mutations per contiguous read over time points (mean ± s.e.m., n = 3 independent experiments). c, Sequencing read alignment of a unique barcoded target detected over three time points. Dendrograms showing the hierarchical relationship of the sequencing reads across time points are on the left of each alignment. A, Red; T, Green; C, Blue; G, yellow. d, Workflow of TRACE MEK1 inhibitor-resistance screen. e, Fold enrichment of mutations across MEK1 cDNA sequence in selumetinib-treated (left) and trametinib-treated (right) samples. Dashed line, fivefold enrichment. f, Summary of identified mutations in TRACE MEK1 inhibitor-resistance screen. g, MAPK–ERK signaling activity as measured by luciferase SRE reporter activity for E38K, V211D and E38KV211D mutants (mean ± s.e.m., n = 3 independent experiments).