Fig. 2

Extending the guide RNA with a matching 5′ end G nucleotide is much more detrimental their activities than extending with a mismatching one in the case of eSpCas9 and SpCas9-HF1. Effect of 5′ extension of the sgRNA with a a mismatching G or b a matching G nucleotide on the activities of SpCas9 nucleases in comparison with using perfectly matching 20-nucleotide-long spacers (data used are from Additional file 1: Figure S2a, c and S2b, d; sites targeted are provided in Additional file 2). Schematics for the spacers used are depicted below the categories as green combs and the 21st G nucleotide extensions are depicted as a red bent end tooth if mismatching; lower case g represents appended nucleotides; numbering corresponds to the distance from the PAM. Tukey-type notched boxplots by BoxPlotR [67]: center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches indicate the 95% confidence intervals for medians; crosses represent sample means; data points are plotted as open circles and correspond to the different targets tested (in total 26 and 10, respectively, for a and b). Each pair of means is statistically different at the p < 0.05 level: a SpCas9–eSpCas9 (<0.001), SpCas9–SpCas9-HF1 (<0.001), SpCas9-HF1–eSpCas9 (<0.015); b statistically different pairs SpCas9–eSpCas9 (<0.001), SpCas9–SpCas9-HF1 (<0.001)