Fig. 2: Variations in the efficiency of single-base tiled crRNAs reveal key determinants of potency beyond target accessibility.
a, Schematic illustrating mCherry RNA regions covered by three-nucleotide-resolution tiled crRNAs. Quantification of silencing efficiency with tiled crRNAs targeting the mCherry regions surrounding crRNA 12 (left) and crRNA 16 (right). Data points in the graph are averages of the normalized mean fluorescence from four representative fields of view per experiment imaged in n = 4 replicates. The data are represented in AU. Errors are the s.e.m. and P values of a one-way ANOVA are indicated (95% confidence interval). b, Schematic showing the sequence of mCherry RNA covered by 61 single-nucleotide tiled crRNAs around the targeted region of crRNA 12. The graph quantifies the silencing efficiency obtained with 61 tiled crRNAs in HEK 293T cells. Data points in the graph are the normalized mean fluorescence from four representative fields of view imaged in n = 2 replicates. UTR, untranslated region. The data are represented in AU. Errors are the s.d. with the 95% confidence interval. c, eGFP fluorescence reporter assay using 41 single-nucleotide tiled crRNAs targeting BCR-ABL1(P190)-eGFP mRNA. The BCR-ABL1(P190)-eGFP mRNA was ectopically expressed in HEK 293T cells using plasmid transfection. This screen with single-base tiled crRNAs aimed to uncover the relationship linking RNA sequence, accessibility and PspCas13b silencing efficiency. The schematic shows the sequence of BCR-ABL1 mRNA covered by 41 tiled crRNAs and the RNA–RNA duplex formed by spacer–target interaction. Data points in the graph are the normalized mean fluorescence from four representative fields of view imaged in n = 3 replicates. The data are represented in AU. Errors are the s.d. with the 95% confidence interval.
