Fig. 3

Fig. 3

a Diagram illustrating one crRNA with NGH PAM (blue) and three crRNAs with NAN PAM (green) targeting wrmScarlet. The sequences of the crRNAs and PAMs are shown below. b Three distinct concentrations of SpG RNP, with the anti-wrmScarlet NGH gRNA 1, were tested in a strain expressing gtbp-1::wrmScarlet. Editing efficiency is defined as the number of F₁ worms exhibiting loss of fluorescence in the F₂ divided by the total number of separated dpy-10 co-edited F₁s. Each dot represents the editing efficiency in each individual P₀ that produced at least ten Dpy or Rol F₁s. All three conditions were carried out in parallel injections (One-way ANOVA followed by Tukey’s test for multiple comparisons p values). c Comparison of editing efficiencies with anti-wrmScarlet NGH gRNA 1 between SpCas9 and SpG, and SpG and SpRY in independent experiments at an RNP concentration of 8.0 µM. Editing efficiency, dots, and numbers are defined as in panel b. Conditions belonging to parallel injections are separated by a dashed line (Student’s t test p value). d In vitro analysis of three anti-wrmScarlet NAN gRNAs. Different RNP combinations were tested in vitro at 37 °C by incubating the RNPs with wrmScarlet PCR product. Top row of bands shows uncleaved PCR products and the specific cleavage products for each gRNA are specified in the figure. The gRNA appears as a faint band at approximately 100 bp. This experiment was performed once. e In vivo analysis of the three anti-wrmScarlet NAN gRNAs. A titration of three distinct SpRY RNP concentrations was performed for gRNAs 1 and 3, while gRNA 2 was tested at 8.0 µM only. The editing efficiency of SpCas9 in NAN sites was evaluated using gRNAs 2 and 3. Editing efficiency, dots, and numbers are defined in panel b. A dashed line separates conditions belonging to parallel injections (One-way ANOVA followed by Tukey’s test for multiple comparisons [NAN 1 and 3], Student’s t test [NAN 2] p values). The percentage of alleles edited in panels b, c, and e was calculated as previously described in Fig. 1i.