Figure 5

hRNA protection at endogenous loci. (A) Introduction of 1 nt substitution in the MSH6 DNA MMR gene that results in a motif recognized by the Type II restriction endonuclease NaeI. Top: the wild-type sequence with single-letter amino acid code. The PAM is indicated with an orange line. Middle: one nucleotide change (in blue) changing the encoded amino acid from proline to alanine. In addition, an NaeI site is created (indicated with a black line). Bottom: additional, silent mutations have been instructed to disrupt the protospacer. Note that PAM disruption in this case would have disrupted the NaeI site. (B) Three different transfections were performed for each mutation: gRNA + template instructing a single nucleotide change (SNS), gRNA + template instructing SNS and additional Cas9 target site disrupting (TSD) mutations, or gRNA + hRNA + template instructing SNS. (C) Experimental workflow. Quantification of the reaction products was performed using the Caliper GX. (D) HideRNAs can be used to protect mutations generated at endogenous loci. On the left, the gene name and intended codon substitution is shown. For each mutation, 4 sequences are shown: the wildtype genomic CRISPR-Cas9 target sequence (WT), the sequence where the SNS has been introduced (marked with a blue ‘M’), the sequence with the SNS plus additional target site disruption (marked with red ‘TSD’), the hRNA spacer sequence. Dots denote sequence identity; dashes indicate that this sequence is not part of the hRNA protospacer. The scores are calculated based on the percentages of digested DNA. Amounts of cut and uncut DNA were quantified on the labchip caliper GX, and the fraction of DNA that contained a Type II restriction site was calculated as: (cut DNA/all DNA)*100%. The experiment was performed three times. Within each replicate, a ‘max-normalization’ was performed—fraction of DNA was divided by the value of the sample with the largest amount of digested DNA. Max normalizations for all replicates were averaged and shown with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Welch one-sided test comparison with ‘M’ (no protection) condition, unless otherwise indicated).