Extended Data Fig. 6: 5′ G-rich motif enhances crRNA abundance and PspCas13b cleavage activity.
(a-d) RT-qPCR analysis to assess the expression of WT crRNAs, crRNAs with an extra G at their 5’ end (31-mer spacer), crRNAs with the first spacer nucleotide substituted to a G (30-mer spacer), and crRNAs with the first and second spacer nucleotides substituted to GG (30-mer spacer) for (a) crRNA40, (b) crRNA39, (c) crRNA26 and (d) crRNA27. crRNAs expression was measured 48 h post-transfection in HEK 293 T cells, N = 3. Data are normalized means and errors are SEM; Results are analysed with one-way Anova test with p-value indicated (95% confidence interval). (e) Averaged expression (from a-d) of unmodified or modified crRNAs harbouring G-rich 5’end. Data are normalized means and errors are SEM; (f) Quality control analysis of in-vitro transcribed crRNAs using TapeStation (left) and 15% Urea PAGE electrophoresis (right); N = 2. (g) In vitro cleavage of PspCas13b incubated with crRNA39 with a 5’ GG motif against a 5’ 6-FAM labelled 100-nt target ssRNA at 2 h timepoint (see uncropped gels in the Source file); N = 1. N is the number of independent biological replicates. Source data are provided as a Source data file.
