Extended Data Fig. 3: CaRPool-seq enables efficient bcgRNA capture and specific target RNA knockdown.
From: Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq
a) Representative BioAnalyzer traces of cDNA and four jointly assayed modalities (GEX = gene expression, bcgRNA = barcode guide RNA, ADT = antibody derived tags, HTO = hashtag oligonucleotides). b) Stacked violin plot showing normalized bcgRNA UMI counts for cells grouped by assigned CRISPR array [total cells n = 9,355, cells with single bcgRNA n = 6,986, (74.7%), n = 29 single bcgRNA conditions; median number of cells 269 per condition, s.d. 97 cells]. c) Bar plots depicting CD46-APC, CD55-FITC, and CD71-PE signal upon Cas13d-mediated knockdown with three alternative gRNAs per target gene relative to the mean of three NT controls measured by flow cytometry. Y-axis shows the mean fluorescent intensity (MFI) relative to the average of all NT cell populations. Two-sided t-test with * p < 0.05, ** p < 0.01, and *** p < 0.001. (N = 3 replicate experiments; error bars depict SEM). Guide RNA g1 was used in CaRPool-seq experiments. Guide RNAs g2 and g3 are used in figures (d) and (e). d) Density plots showing the CD46-APC, CD55-FITC, and CD71-PE signal upon Cas13d-mediated knockdown with either 1, 2, or 3 copies of the same gRNA (g1) per CRISPR array or 2 and 3 alternative gRNAs (g2, g3). Vertical lines mark the threshold (2nd percentile of combined NT conditions) for CD-protein negative cells, indicating the percent negative cells for one replicate experiment. N > 5000 cells examined per sample. Shown is one representative replicate. e) Summary analysis of biological replicate experiments (n = 3) as shown in (d). Y-axis shows the mean fluorescent intensity (MFI) relative to the average of all NT cell populations. The Analysis suggests that target gene knockdown differences between the number of gRNAs per array are more pronounced than differences between gRNA identities with the same total gRNA count, given that gRNA efficiencies are comparable as shown in (c). CRISPR arrays encoding multiple gRNAs against the same target may be used to further enhance target knockdown. Two-sided t-test with * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars depict SEM. f) Scatterplots showing normalized pseudobulk RNA UMI count profiles of cells grouped by indicated CRISPR arrays (y-axis) and control cells that received non-targeting (NT) gRNAs (x-axis). Respective target genes (CD46, CD55, CD71) are highlighted in color. Genes on the MT chromosome are colored green. Other significantly differentially regulated genes (Wilcoxon’s rank sum test; adjusted p-value < 0.05) are highlighted in black. Genes highlighted in black showed a median expression change of 12.8% (s.d. 9.9%). CD71 + CD71 was not included in the experiment. g) Volcano plots showing differential gene expression results cells grouped by indicated CRISPR arrays and control NT cells. Cells grouping is the same as in (f). The x-axis indicates log-transformed fold changes. The y-axis depicts -log10-transformed adjusted p-values (Wilcoxon’s rank sum test). Significantly differentially regulated genes (adjusted p-value < 0.05) are highlighted in red.
