Fig. 1: High-throughput design and evaluation of a TP53 prime editing sensor library.
From: High-throughput evaluation of genetic variants with prime editing sensor libraries
a, Schematic of our overall approach. We aim to engineer variants observed in patients with high throughput to perform functional screens in diverse contexts, elucidating variant functions to improve our ability to stratify and treat patients. b, Schematic of the sensor framework, which links each pegRNA to its editing outcome at the endogenous locus. c, We used PEGG to generate a TP53 prime editing sensor library targeting >1,000 cancer-associated TP53 variants with a median of 30 pegRNAs per variant. d, Heatmap visualization of the pegRNAs included in the TP53 sensor library, which includes SNVs, indels and silent substitutions. e, Correlation between editing at the sensor and endogenous locus in eight TP53-targeting pegRNA-sensor pairs at day 3 (D3) and day 7 (D7) posttransduction. f, Schematic of the screening protocol. The prime editing sensor library is transduced into cells constitutively expressing PEmax, and screening is performed in the presence or absence of the small molecule Nutlin-3. g, The average correct editing percentage among all pegRNAs in the library (left) or when considering only the most efficient pegRNA for each variant (right) at various timepoints in both conditions for pegRNA-sensor pairs with at least 100 sequencing reads; n = 3 biologically independent replicates. Data are presented as mean values with a 95% confidence interval. h, Rank plot of the correct editing percentage of the most efficient pegRNA per variant, as assayed at the sensor locus, at each timepoint. Source data and code to reproduce this figure can be found at https://github.com/samgould2/p53-prime-editing-sensor/blob/main/figure1.ipynb. AD1/2, activation domain 1/2; BlastR, blasticidin selection marker; CTD, C-terminal domain; DEL, deletions; EIF1α, eukaryotic initiation factor 1 alpha; INS, insertions; nCas9, nicking Cas9; NLS, nuclear localization signal; PRR, proline rich region; Puro, puromycin selection marker; P2A, peptide 2ART, reverse transcriptase; STOP, U6 polyT termination sequence; tevo, tevopreQ1; U6, U6 promoter.
