Supplementary Figure 2: Additional analysis of large-scale mismatched sgRNA screen. | Nature Biotechnology

Supplementary Figure 2: Additional analysis of large-scale mismatched sgRNA screen.

From: Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs

Supplementary Figure 2

(a, b) Comparison of growth phenotypes (γ) of all sgRNAs derived from replicates of the (a) K562 (n = 119,201 sgRNAs) and (b) Jurkat screens (n = 119,229 sgRNAs). Marginal distributions are normalized to element count in each category. r2 = squared Pearson correlation coefficient for targeting sgRNAs (mismatched and original). (c) Comparison of γ of perfectly matched sgRNAs from the K562 screen in this work and a previously published K562 screen14 (average of two replicate screens). n = 4,830 sgRNAs; r2 = squared Pearson correlation coefficient. (d) Comparison of γ of perfectly matched sgRNAs in K562 and Jurkat cells reveals substantial differences, likely reflecting cell-type specific gene essentiality (average of two replicate screens). n = 4,892 sgRNAs; r2 = squared Pearson correlation coefficient. (e) Comparison of mismatched sgRNA relative activities in K562 and Jurkat cells, classified by the difference in γ of the corresponding original guide. n = 15,103 (left) and 26,409 (right) sgRNAs; r2 = squared Pearson correlation coefficient. (f) Distribution of mismatched sgRNA relative activities for sgRNAs with 1 mismatch (left) or 2 mismatches (right). (g) Distribution of mismatched sgRNA relative activities stratified by sgRNA GC content, grouped by mismatches located in positions –19 to –13 (PAM-distal region), positions –12 to –9 (intermediate region), and positions –8 to –1 (PAM-proximal/seed region). n = 282-7,592 sgRNAs. (h) Distribution of mismatched sgRNA relative activities stratified by the identity of the 2 bases flanking the mismatch, grouped by mismatches located in the three regions as in g. n = 155-2,031 sgRNAs. (i) Distribution of mismatched sgRNA relative activities stratified based on whether or not the invariant first G of the sgRNA (position –20) matches the genome, grouped by mismatches located in the three regions as in g. n = 4,267-11,524 sgRNAs. (j) Comparison of mean CRISPRi relative activities from large-scale screen and cutting frequency determination (CFD) scores27. Values are compared for identical combinations of mismatch type and mismatch position; mean relative activities were calculated by averaging relative activities for all mismatched sgRNAs with a given combination. n = 228 mismatch type/position combinations; r2 = squared Pearson correlation coefficient. (k) Distribution of sgRNA series by number of sgRNAs with intermediate activity (0.1 < relative activity < 0.9), using only sgRNAs with a single mismatch (top) or all mismatched sgRNAs (bottom). Lines in violin plots in panels g, h, i denote distribution quartiles.

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