Fig. 5: crisprDesign workflow to design gRNAs tiling CD55 and CD46 using CasRx.

On the left: schematic showing the major steps involved in designing CasRx gRNAs targeting CD55 and CD46. Two inputs are required: mRNA sequences of CD55 and CD46 and a CrisprNuclease object from crisprBase. a Relationship between on-target CasRx-RF score calculated in crisprScore and LFCs from the pooled FACS tiling CasRx screening data (see Methods). A higher LFC indicates higher gRNA activity. b Relationship between LFCs from the CasRx screening data and gRNA context for CD46 and CD55: gRNAs targeting 5\({}^{\prime}\) UTR and 3\({}^{\prime}\) UTR for the canonical transcript, and guides targeting a low and high number of isoforms for each of the genes. gRNAs targeting more isoforms show higher enrichment in the screening data. The boxes represent the 25−75% interquartile ranges (IQR), and the central lines represent the median values. The whiskers extend 1.5 times the IQR from the median value. The number of data points for each boxplot is specified above the whiskers. The full isoform annotation is stored in the GuideSet objects. c Left: relationship between observed LFCs of on-target gRNAs in the CD55 screen and predicted LFCs of single-mismatch gRNAs using the off-target CFD-CasRx score implemented in crisprScore (see Methods). Right: same as left, but for double-mismatch gRNAs. d gRNAs selected in the CD46 screen for high on-target activity (CasRx-RF score) and targeting a common exon across all protein-coding isoforms enrich for high gRNA activity. Source data are provided as a Source Data file.