Fig. 4: Indel frequency of gRNAs at targets with different PAM sequences in HEK293T cells.
From: CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context
a Heatmap of indel frequencies (Dox- cells) of four gRNAs targeting sites with 5′-N−1GGN+1-3′ PAMs clustered by Euclidean distance (missing values were linearly interpolated from targets with the same N−1N+1 context). Averages of columns and rows are indicated. Leaves in the left dendrogram are coloured as in Fig. 3b. b Pearson’s correlation between indel frequency of four gRNAs and their specificity extended with local sliding (CRISPRspecExt, blue colour). *p < 0.05, **p < 0.01, ns non-significant (gRNA PAX5 p = 7.05E-03, ADRA2C p = 0.02, CHRNB2 p = 0.11, BMP3 p = 8.73E-3). The specificity score CRISPRspec, not influenced by PAM contexts, is indicated with a dashed line. c Indel frequency (Dox- cells) of gRNAs binding at targets with different PAM binding sites (X-axis). The swarm plot details the indel frequencies of each gRNA. Boxes represent the first and third quartiles (Q1 and Q3). The median is shown as a line; whiskers extend up (or down) to the last (or first) value lower (or greater) than Q3+1.5*(Q3-Q1) (or Q1-1.5*(Q3-Q1)). Number of gRNAs per box, left to right: 59; 56; 53; 56; 60; 54; 62; 60; 58; 61; 59; 62; 58; 57; 62; 63. d Indel frequency (Dox+ cells) at targets with alternative PAMs but canonical GG local sliding PAM upstream (top) or downstream (bottom). Boxes and swarm plots are defined as in (c). Binding contexts represented in less than three of four gRNAs or with G+1 (top) or G−1 (bottom) were excluded. See Supplementary Figs. 12–15 for results on all PAMs and cell treatments. One-sided t-test p values from left to right, top to bottom: 0.17; 9.72E-04; 1.69E-07; 0.94; 1.40E-03; 2.59E-07 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 1E-04). The alternative hypothesis is that efficiencies at targets with G−1 (top) or G+1 (bottom) have a larger mean than, respectively, H−1 or H+1. Number of elements per box, from left to right, top to bottom: 30; 12; 35; 11; 36; 11; 35; 12; 36; 11; 35; 12. e Heatmap of indel frequencies (Dox+ cells) of four gRNAs at targets with 5′-N-1AGN+1-3′ PAMs. See (a) for further details. Source data are provided as a Source Data file.
