a) Pooled screens detect changes in gRNA abundance among bulk populations of cells, which limits them to simple readouts based on cell frequencies. b) Arrayed screens support complex readouts such as transcriptome profiling, but cells transduced with different gRNAs have to be physically separated. c) CROP-seq uses droplet-based single-cell RNA-seq to profile each cell’s transcriptome together with the expressed gRNA, and knockout signatures are derived by averaging across cells that express gRNAs for the same target gene. d) Data analysis identifies pathway signature genes and quantifies the effect of specific gRNAs on these signatures. e) The CROP-seq lentiviral construct includes a gRNA cassette within the 3’ long terminal repeat (LTR), which is duplicated during viral integration. It expresses an RNA polymerase III transcript for genome editing and a polyadenylated RNA polymerase II transcript detected by single-cell RNA-seq. f) Cloning the hU6-gRNA cassette into the 3’ LTR to generate CROPseq-Guide-Puro does not compromise lentiviral function for gRNAs. In contrast, 1,885 bp of filler DNA result in a 98-fold reduction of the viral titer. g) Genome editing efficiencies and indel signatures are highly similar between LentiGuide-Puro and CROPseq-Guide-Puro. h) CROP-seq can detect gRNAs from single-cell transcriptomes. i) The rate of successful gRNA assignments is associated with single-cell transcriptome quality, expressed as the number of detected genes per cell. Most cells were assigned to one gRNA, except for a small fraction of cell doublets. Error bars, 95% CI. j) Performance statistics across all CROP-seq experiments.