Fig. 5: Functional validation of pathogenic TP53 variants identified with prime editing screening.
From: High-throughput evaluation of genetic variants with prime editing sensor libraries
a, LFC of 29 pegRNAs selected for validation at D34 in the Nutlin-treated condition. Variants with insufficient control counts (I195T, E285K, G325A, Y327Ter, A347D) are represented by LFC = 0. b, Schematic of the competition assay methodology. A549-PEmax cells are transduced with individual pegRNAs with an mScarlet fluorescent marker. After 7–10 days, these A549-PEmax-pegRNA cells are mixed with parental, uncolored A549-PEmax cells and split into untreated or Nutlin-treated conditions. Flow readouts of the RFP+ cell fraction are then taken at several timepoints. c, Selected representative competition assays show the change in the RFP+ cell fraction in the presence (orange) or absence (blue) of Nutlin-3. Data are presented as mean values at each timepoint, with a 95% confidence interval. d, ∆RFP% from the D0 to the D7 and D4 timepoints for each variant in the presence or absence of Nutlin-3; n = 3 biologically independent replicates performed per condition. Data are presented as mean values with a 95% confidence interval. e, Scatterplot of the LFC of Nutlin-treated pegRNAs at D34 of the screen, and the corresponding ∆RFP% at D7 in the competition assay. Points colored by endogenous editing percentage, with ‘X’ indicating no endogenous editing data. f, Same as e but for the untreated condition. g, Scatterplot of sensor editing percentage at D16 and the corresponding endogenous editing percentage of pure A549-PEmax-pegRNA cell lines for pegRNAs included in competition assays. Source data and code to reproduce this figure can be found at https://github.com/samgould2/p53-prime-editing-sensor/blob/main/figure5.ipynb. rp, Pearson correlation; rs, Spearman correlation.
