Figure 2
From: HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing
Protection against re-cutting by hRNAs. (A) Upper part: Cas9 in complex with a full-length gRNA (represented as red, curving line) can still induce a break despite the presence of a spacer-protospacer mismatch (blue rectangle). Lower part: a gRNA with truncated protospacer that has complete homology to the target sequence from the top part stably binds this sequence, but does not induce a break. It hides the target site from Cas9 bound to a full-length gRNA. (B) hRNAs with matching, truncated protospacer sequence protect the modified reporter sequence from re-cutting. hRNAs 1 and 2 use the original PAM (upper part); hRNAs 3 and 4 use an adjacent PAM. The start codon is marked green/red. Note that the sequence of the hRNA protospacers allows hybridization to the T of the ATG, (marked in green), whereas a T-U mismatch is formed with the protospacer of the gRNA (indicated in light grey). (C) Experimental setup for evaluating the re-cutting prevention capability of hRNAs. A vector expressing a hRNA and a gRNA is combined with an ATG-inducing ssODN. (D) GFP percentages obtained with 6 different conditions: gRNA + re-cutting-sensitive (ATG) or -protected (ATG + PAM) template; gRNA + hRNA 1–4 plus re-cutting sensitive (ATG) template. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Welch one-sided test comparison with ‘ATG ssODN’ condition, color indicates for which condition this applies). Error bars denote standard error of mean (n = 3). (E) The protection score was calculated as follows: (Gi − Gsens)/(GPAM − Gsens), with Gi the percentage of GFP positive cells for a sample, Gsens the percentage of GFP positive cells obtained with the gRNA and the re-cutting sensitive template, and GPAM the percentage of GFP positive cells obtained with the gRNA and the re-cutting-protected template. Colors of each bar as in (D).
