Fig. 1: The efficiency of PspCas13b silencing is highly variable between crRNAs.
a, Schematic of PspCas13b silencing assay used to monitor mCherry RNA target recognition and degradation. b, Representative fluorescence microscopy images show the silencing of mCherry transcripts with a potent or ineffective targeting crRNA versus an NT control crRNA in HEK 293T cells (n = 3). c, Schematic of the 16 PspCas13b crRNAs targeting various regions of mCherry RNA. The detailed spacer and direct repeat regions of a representative crRNA are indicated on the right. d, crRNA dose-dependent silencing of mCherry transcripts with either NT (orange) or targeting (gray) crRNAs. Data points in the graph are averages of the normalized mean fluorescence from four representative fields of view imaged in n = 3 or 4 replicates (crRNAs 1, 4, 8 and 9 and 14, n = 4; crRNAs NT, 2, 3, 5, 6, 7, 10, 11, 12, 13, 15 and 16, n = 3). The data are represented in arbitrary units (AU). Errors are the s.e.m. and P values of a one-way ANOVA are indicated (95% confidence interval). NS, not significant. e, Silencing of mCherry transcripts with active PspCas13b, dPspCas13b or crRNA only (no PspCas13b) and either NT crRNA or crRNA 12. Data points in the graph are averages of 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.e.m. and P values of an unpaired two-tailed Student’s t-test are indicated (95% confidence interval). f, RT–qPCR assays measuring the silencing efficiency of either NT crRNA or crRNA 12 by active PspCas13b, dPspCas13b or crRNA only (n = 3). Data are the normalized means and errors are the s.e.m. Results were analyzed by an unpaired two-tailed Student’s t-test and P values are indicated (95% confidence interval). g, Western blot analysis examining the expression level of active PspCas13b or dPspCas13b and mCherry proteins in HEK 293T cells expressing either crRNA 12 or NT crRNA (n = 3).
