Supplemental data on activity of RNF183, FAM86B2, and SUGP1 crRNAs. (A) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with VBC-derived (V) RNF183 crRNAs. (B) Incomplete targeting with the crRNF183 pool may be associated with an overlapping target region and/or spanning of a common polymorphism (rs3750534) within a target sequence for crRNF183 (V) #3 and crRNF183 (V) #4, which have particularly low activity. (C) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with the VBC-derived (V) crFAM86B2 crRNA pool. Insufficient targeting of the crFAM86B2 pool may be due to all four crRNAs targeting overlapping regions, and therefore competing with each other. (D) Phenotyping of individual and pooled SUGP1 crRNAs. Heatmap displaying quantification of SUGP1 protein signal and localization ratio (perinuclear versus peripheral) of GFP-BICD2N-FRB and PTS-RFP-FKBP spots in U-2 OS PEX cells with the indicated treatments. “(D)” and “(V)” indicate source of crRNA design (D, Discovery; V, VBC score). Data points represent mean per cell values aggregated at well levels (minimum of 100 cells analyzed per condition; four wells per condition). Color scale of individual features was adjusted based on minimum and maximum raw values. The guides selected for RNA-seq are labeled in gold text. (E–G) Quality control for samples submitted for RNA-seq. (E) Violin plots of intensity of SUGP1 protein signal at the single cell level (median, bold line; first/third quartile, dashed lines; minimum of 100 cells analyzed per condition). (F and G) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with crSUGP1 #1 (F) or crXCR1 #1 (G). In A, C, F, and G, bar graphs display mean ± SD, and the efficiency scores are out of 100. Corresponding sequences from cells transfected with NTC crRNAs #1–4 (A and C) and NTC crRNA #1 (F and G) were used as a reference.