Fig. 2.

Off-target editing with a more efficient Editase can be reduced using a guide with a site-specific A–G mismatch to Mecp2 mRNA. (A) Percent A-to-I editing at the R106Q (mean ± SD, n = 3) site after transfection into N2A cells of guide RNA and Editase^WT or Editase^E488Q (mean ± SD; n = 3, includes data in B). (B) Representative chromatograms of Mecp2^R106Q cDNA edited with Editase^WT (Top) or Editase^E488Q (Bottom). (C) Mecp2 mRNA relative to two different guide RNAs (green). The standard guide (Top) contains an A–C mismatch (R106Q) at the target A (highlighted in bold) to enhance editing. The modified guide (Bottom) contains an A–G mismatch at an off-target A marked by an asterisk to inhibit editing at this site. (D) Chromatograms of Mecp2 cDNA after transfection of N2A cells with Editase^E488Q and a guide containing only the mismatch at the target site (Top) or the modified guide containing both the on-target A–C mismatch and the A–G mismatch at the off-target site (Bottom). (E) Off-target editing is severely reduced with the guide containing the A–G mismatch (mean ± SD; n = 3, includes data in D). (F) Presence of the off-target A–G mismatch does not affect editing at the R106Q site (mean ± SD; n = 3, includes data in D). Light-gray bars, cells transfected with Editase alone; dark-gray bars, cells transfected with Editase and guide; black bars, cells transfected with Editase and guide containing the A–G mismatch. **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test. ns, not significant.