Fig. 2: Concatenated Cas13d gRNAs show sequence-dependent interference which can be overcome by gRNA expression from separate promoters.

A Overview of the pooled Cas13d screening pipeline. B Schematic of the dual gRNA concatenation strategy U6-g1-g2. mU6 = mouse U6 promoter, diamonds = direct repeat DR36, squares = target-specific spacer sequence. Cas13d can process concatenated gRNA arrays into single gRNAs. C Correlation between tau values from two technical screen replicates, following 19 days of imatinib treatment using the U6-g1-g2 strategy. Pearson correlation was used to determine the r value. D Correlation between tau values from gRNA-gNTC and gNTC-gRNA combinations following 19 days of imatinib treatment using the U6-g1-g2 strategy. Pearson correlation was used to determine the r value. E Bar chart of the tau values from the three best-performing gRNA targeting PTPN1 and SOS1 in combination with 30 gNTCs in both orientations: gRNA-gNTC (top) and gNTC-gRNA (bottom). F Schematic of the dual promoter gRNA expression strategy mU6-g1-hU6-g2. mU6 = mouse U6 promoter, hU6 = human U6 promoter, diamonds = direct repeat DR36, squares = target-specific spacer sequence. G Correlation between tau values from two independent screen replicates, following 19 days of imatinib treatment using the U6-g1-U6-g2 strategy. Pearson correlation was used to determine the r value. H Correlation between tau values from gRNA-gNTC and gNTC-gRNA combinations following 19 days of imatinib treatment using the U6-g1-U6-g2 strategy. Pearson correlation was used to determine the r value. I Schematic of the single-gene array. J Flow cytometric quantification of CD46, CD47, CD71, and CD63 residual protein levels in cells expressing either a single-gene array or one of the three single gRNAs that were concatenated in the single-gene array. Values represent the mean of biological replicates; error bars, SD (n = 3). K Schematic of the dual promoter single-gene array expression strategy used for subsequent U6-a1-U6-a2 libraries. mU6 = mouse U6 promoter, hU6 = human U6 promoter, diamonds = direct repeat DR36, squares = target-specific spacer sequence. Source data are provided as a Source Data file.