Extended Data Fig. 4: Evaluation of Cas13d specific off-target effects in CaRPool-seq.

a) Sites, and relative expression levels of gRNA-dependent predicted off-target transcripts from gRNAs targeting CD46, CD55 and CD71. Red letters indicate mismatches and indels to cognate perfect match target site. E-values derived from Blastn. (Wilcoxon’s rank sum test * p.adj. < 0.05, ** p.adj. < 0.01, *** p.adj. < 0.001). b) Bulk RNA-seq result for Cas13d, Cas9-nuclease, and KRAB-dCas9-MeCP2 based targeting of CD55 using three independent CD55-targeting and NT (s)gRNAs, respectively. Volcano plots show differential gene expression results of CD55 targeting conditions relative to corresponding NT conditions grouped by indicated CRISPR effector protein. The x-axis indicates log-transformed fold changes. The y-axis depicts -log10-transformed adjusted p-values (DESeq2). Significant differentially regulated genes (adjusted p-value < 0.05, Wilcoxon’s rank sum test) are highlighted in red. The three approaches show a varying number of differentially expressed genes in addition to CD55 reduction (n = 1 Cas13d, n = 3 Cas9, n = 30 KRAB-dCas9-MeCP2). Cas13d gRNA and Cas9 sgRNA efficiency is shown in Extended Data Fig. 3c and Extended Data Fig. 5a. c) Differential gene expression of mitochondrial genes. Differential gene expression was assessed between all 26 cell populations expressing gene-targeting gRNAs and cells expressing a single NT gRNA. Across all differential gene expression analyses (n = 26), none of the 13 mitochondrial genes encoded on the mitochondrial chromosome was expressed significantly different from control cells (adjusted p-value < 0.05; Wilcoxon’s rank sum test). The figure shows the average log2 fold change (FC) across all 13 mitochondrial genes per condition (n = 26) grouped by the number of target genes (left) and number of targeting gRNAs (right) indicating that observed changes are independent of target RNA and gRNA amounts. d) Fraction of cells classified into indicated cell cycle stages for each condition (n = 29). Dotted lines indicate the means of the three NT cell populations. Statistical testing for differences in cell cycle stage assignment for cell populations targeting e) varying numbers of target genes per cell, or using f) increasing numbers of gRNAs per cell did not show significant differences to NT control cell populations expressing zero targeting gRNAs (p-values derived from two-sided students t-test, not corrected for multiple testing. Bars in (e) and (f) show mean. Error bars depict SEM. N numbers indicated inside bars).