Fig. 3

Construction of a barcoded library for inducible guide RNA expression. a Schematic of the guide RNA expression library. The RPR1 promoter is regulated by two tetO operator sites. In the presence of tetracycline, this promoter is de-repressed and drives expression of a variable guide RNA sequence in a constant sgRNA scaffold. A random nucleotide barcode with an adjacent T7 RNA polymerase promoter is embedded elsewhere in the plasmid. b Schematic of the process for generating the guide RNA library. Guides are cloned first, and then barcodes are added in a transformation with controlled diversity. c Distribution of barcode-to-guide assignment results, illustrating the high frequency of errors in cloned guide RNAs. d Cumulative distribution of the number of barcodes assigned to each guide RNA