Fig. 4: Incorporation of a 5′ G-rich motif enhances the potency of ineffective crRNAs.
a–g, Incorporation of a G-rich motif at the 5′ end of ineffective spacer sequences (crRNA39 (a), crRNA40 (b), crRNA24 (c), crRNA25 (d), crRNA26 (e), crRNA27 (f) and crRNA28 (g)) targeting mCherry through the insertion or substitution of a G base greatly enhanced their silencing efficiency. The red color in the spacer sequence indicates nucleotide insertion or substitution. Data points in the graph represent the averaged mean fluorescence from four representative fields of view per condition imaged (a,b, n = 3; c–g, n = 4). WT, wild type. 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). h, Schematic of PspCas13b silencing assay performed in HEK 293T cells using IVT or chemically synthesized crRNAs with or without a 5′ G-G motif. i,j, Assessment of the silencing efficiency of IVT (i) and chemically synthesized (j) crRNA pairs that either fully base pair with the target (WT) or harbor a mismatched 5′ G-G motif in their spacer sequence. The silencing efficiency was monitored at the 24-h and 48-h time points after transfection of crRNAs. Data points in the graph are the averaged mean fluorescence from four representative fields of view per imaged condition (n = 3). 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). k, Gel electrophoresis showing the expression profile of purified recombinant PspCas13b (n = 3). l, In vitro cleavage of a 100-nucleotide ssRNA (labeled with a 5′ 6-FAM probe) using recombinant PspCas13b loaded with crRNA 39 with or without a 5′ G-G motif at the 0-h, 2-h and 4-h time points (n = 1). m, In vitro RNA cleavage obtained with PspCas13b incubated with crRNA with or without a 5′ G-G motif and a target ssRNA labeled with 5′ 6-FAM. The arrows indicate cleaved RNA products. The cleavage was assessed at the 0-h and 2-h time points (n = 3).
