Fig. 3: Multiplexed prime editing screen targeting premature stop codons to essential genes induces negative growth phenotypes.
From: A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening
a, Schematic of design pipeline for generating StopPR epegRNA library. CRISPick58 and gene annotations used to identify edits. ‘Inaccessible’ filters removed nonsense or spacer- and codon-matched synonymous edits that could not be made with prime editing (for example, if edit occurred upstream of the Cas9 H840A nick). b, Schematic illustrating the intended consequences of prime editing for each epegRNA category in StopPR. Intended edit is shown in red. c, StopPR composition, including numbers of genes and/or protospacers targeted, and numbers of stop epegRNAs with different substitutions, positions and codons, for each edit length. Multiple codons were often targeted near the same protospacer, with 7,785 protospacers targeting 16,991 codons. d, Growth phenotypes for epegRNAs from independent biological replicates of StopPR screen collected 14 days post transduction. Dotted lines denote phenotype cutoffs (Z < −2). Correlation (Pearson’s r) between replicates indicated for each epegRNA category. Density plots on top and side show data distribution per epegRNA category for replicates 1 and 2, respectively. e, Gene-level growth phenotypes from StopPR screen (calculated as average phenotype of the absolute strongest two stop epegRNAs per gene on day 14 post transduction) binned by CRISPRi phenotypes (as previously determined in K562 cells59, split into three equally sized bins with number of genes denoted as n). Individual P values from one-way ANOVA (F = 59.49) and two-sided Tukey post hoc denoted. Median and IQR of the full set of epegRNAs used in this analysis for each phenotype bin are indicated. Whiskers extend 1.5× IQR past the upper and lower quartiles. Dotted line denotes phenotype cutoff (Z < −2). f, Growth phenotypes from StopPR screen (y axis, replicate-averaged) and independent retest of individual epegRNAs (x axis, triplicate-averaged), for stop epegRNAs (ten), spacer- and codon-matched synonymous epegRNAs (ten) and negative controls (five). Measurements sampled from day 14 post transduction. g, Percentages of sequencing reads containing precise substitutions from cells sampled 7 days post transduction using same epegRNA constructs as in f. Matched stop (red) and synonymous (blue) epegRNAs targeting the same codon for each site. Exact edits denoted. Data and error bars represent mean ± s.d. (n = 3 independent biological replicates). b, Created with BioRender.com.
