Extended Data Fig. 5: Mutagenesis study reveals the importance of a 5′ G-rich motif for potent silencing activity of crRNA.
(a) Dose-dependent silencing of mCherry transcript with non-targeting crRNA (NT), 11 unmodified crRNAs (WT) that possess a GG sequence at their 5’end (white bars), or the same 11 crRNAs were the 5’ GG sequence of the spacer is mutated to 5’ CC through 1–3 nucleotides mutagenesis. Data points in the graph are normalized mean fluorescence from 4 different fields of view imaged in N = 2. The data are represented in arbitrary units (A.U.). Errors are SD with a 95% confidence interval. (b) Mutagenesis analysis of spacer 1–3 nucleotides (5’ end) examining the impact of C to G substitutions on crRNA silencing efficiency. The nucleotides in red highlight mismatch positions in the spacer sequence. Data points in the graph are averages of mean fluorescence from 4 representative fields of view per condition imaged; N = 3. Errors are SEM and p-values of one-way Anova test are indicated (95% confidence interval). The data are represented in arbitrary units (A.U.). (c) Representative fluorescence microscopy images show the silencing efficiency of the mCherry transcripts with NT, WT and mutant crRNAs in HEK 293 T cells. NT is a non-targeting control crRNA. Scale bar = 400μm. Similar results were obtained in 3 independent experiments in HEK 293 T cells. N is the number of independent biological replicates. Unprocessed representative images are provided in the Source Data file.
